Human monoclonal antibodies to activin receptor-like kinase-1

ABSTRACT

The present invention relates to antibodies including human antibodies and antigen-binding portions thereof that bind to the extracellular domain (ECD) of activin receptor-like kinase-1 (ALK-1) and that function to abrogate the ALK-1/TGF-beta-1/Smad1 signaling pathway. The invention also relates to heavy and light chain immunoglobulins derived from human anti-ALK-1 antibodies and nucleic acid molecules encoding such immunoglobulins. The present invention also relates to methods of making human anti-ALK-1 antibodies, compositions comprising these antibodies and methods of using the antibodies and compositions. The invention also relates to transgenic animals or plants comprising nucleic acid molecules of the present invention.

This application claims priority under 35 U.S.C. §119(e) from U.S.provisional application 60/715,292, filed Sep. 7, 2005, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to human monoclonal antibodies andantigen-binding portions thereof that bind to the extracellular domain(ECD) of activin receptor-like kinase-1 (ALK-1). The invention alsorelates to nucleic acid molecules encoding such antibodies andantigen-binding portions, methods of making human anti-ALK-1 antibodiesand antigen-binding portions, compositions comprising these antibodiesand antigen-binding portions and methods of using the antibodies,antigen-binding portions, and compositions.

BACKGROUND OF THE INVENTION

ALK-1 is a type I cell surface receptor for transforming growth factorbeta receptor type 1 (TGF-beta-1). Human ALK-1 is a 503 amino acidpolypeptide, which includes a signal sequence (amino acids: 1-21), aN-terminal extracellular TGF-beta-1 ligand binding domain or ECD (aminoacids: 22-118), a single transmembrane domain (amino acids: 119-141) aregulatory glycine/serine rich (GS) domain (amino acids: 142-202) and aC-terminal a serine-threonine kinase domain (202-492). The amino acidsequence of human ALK-1 disclosed in Attisano et al. Cell, 1993, vol.75, pp. 671-680 includes Ser at position 172 (Genbank record L17075),while U.S. Pat. No. 6,316,217 claims the amino acid sequence of humanALK-1 with Thr at position 172 (Genbank record NM_(—)000020). ACVRL1gene encoding a full-length human ALK-1 disclosed in Attisano et al. iscommercially available from Invitrogen Inc., Clone ID IOH21048. AlthoughALK-1 shares 60-80% overall homology with other type I receptors (ALK-2through ALK-7), ECD of ALK-1 is remarkably divergent from ECDs of otherALK family members. For example, in human, only ECD of ALK-2 issignificantly related to ECD of ALK-1 (sharing approximately 25% aminoacid identity). U.S. Pat. No. 6,316,217; ten Dijke et al. Oncogene,1993, vol. 8, pp. 2879-2887; Attisano et al. Cell, 1993, vol. 75, pp.671-680.

In general, TGF-beta superfamily ligands exert their biologicalactivities via binding to heteromeric receptor complexes of two types (Iand II) of serine/threonine kinases. Type II receptors areconstitutively active kinases that phosphorylate type I receptor uponligand binding. In turn, activated type I kinases phosphorylatedownstream signaling molecules including the various Smads, whichtranslocate to the nucleus and lead to a transcriptional response.Heldin et al. Nature, 1997, vol. 390, pp. 465-471. In the case of ALK-1,we have shown that Smad1 is specifically phosphorylated and translocatesto the nucleus where it directly regulates the expression of the Smad1responsive genes Id1 and EphB2.

ALK-1 is expressed highly and selectively in endothelial cells and otherhighly vascularized tissues such as placenta or brain. We have shown byAFFYMETRIX® profiling and real-time RT-PCR that the expression of ALK-1in endothelial cells highly exceeds the expression of its co-receptorsactivin type II and endoglin, its ligand TGF-beta-1 or ALK-5. Mutationsin ALK-1 are associated with heredity hemorrhagic telangiectasia (HHT),suggesting a critical role for ALK-1 in the control of blood vesseldevelopment or repair. Abdalla et al. J. Med. Genet., 2003, vol. 40, pp.494-502; Sadick et al. Hematologica/The Hematology J., 2005, vol. 90,818-828. Furthermore, two independent studies of ALK-1 knockout miceprovide the key in vivo evidence for ALK-1 function during angiogenesis.Oh et al. Proc Natl Acad Sci USA, 2000, vol. 97, pp. 2626-2631; Urnesset al. Nature Genetics, 2000, vol. 26, pp. 328-331.

Angiogenesis is the physiological process involving the formation of newblood vessels from pre-existing vessels and/or circulating endothelialstem cells. This is a normal process in growth and development, as wellas in wound healing. However, this is also a fundamental step in thetransition of tumors from a dormant state to a malignant state. Hanahanand Folkman, “Patterns and Emerging Mechanisms of the Angiogenic SwitchDuring Tumorigenesis,” Cell, 86(3):353-364, 1996; Carmeliet,“Angiogenesis in Health and Disease,” Nature Medicine, 9(6):653-660,2003; Bergers and Benjamin, “Tumoreigenesis and the Angiogenic Switch,”Nature Reviews, 3:401-410, 2003. In diseases like cancer, the body losesthe ability to maintain balanced angiogenesis. New blood vessels feeddiseased tissues, destroy normal tissues, and in the case of somecancers, the new vessels can allow tumor cells to escape into thecirculation and lodge in other organs (tumor metastases). Angiogenesisinhibitors, including monoclonal antibodies (mAbs), are a very promisingclass of drugs targeted against this abnormal process to block or slowtumor growth.

In addition to a role in solid tumor growth and metastasis, othernotable conditions with an angiogenic component are, for example,arthritis, psoriasis, neovascular age-related macular degeneration anddiabetic retinopathy. Bonnet et al. “Osteoarthritis, Angiogenesis andInflammation,” Rheumatology, 2005, vol. 44, pp. 7-16; Creamer et al.“Angiogenesis in psoriasis,” Angiogenesis, 2002, vol. 5, pp. 231-236;Clavel et al. “Recent data on the role for angiogenesis in rheumatoidarthritis,” Joint Bone Spine, 2003, vol. 70, pp. 321-326; Anandarajah etal. “Pathogenesis of psoriatic arthritis,” Curr. Opin. Rheumatol., 2004,vol. 16, pp. 338-343; Ng et al. “Targeting angiogenesis, the underlyingdisorder in neovascular age-related macular degeneration,” Can. J.Ophthalmol., 2005, vol. 40, pp. 352-368; Witmer et al. “Vascularendothelial growth factors and angiogenesis in eye disease,” Progress inRetinal & Eye Research, 2003, vol. 22, pp. 1-29; Adamis et al.“Angiogenesis and ophthalmic disease,” Angiogenesis, 1999, vol. 3, pp.9-14.

Anti-angiogenic therapies are expected to be chronic in nature.Accordingly, targets with highly selective endothelial function, such asALK-1, are preferred to reduce attrition resulting from side effects.Furthermore, given the remarkable divergence of the ALK-1 ECD from ECDsof other ALK family members, mAb raised against the human ALK-1 ECD areexpected to selectively target ALK-1. Based on these considerations, amonoclonal antibody against the ALK-1 extracellular domain that mayinhibit dimerization with the type II receptor and therefore block Smad1phosphorylation and the downstream transcriptional response is highlydesirable.

R&D Systems, Inc. makes and sells a monoclonal anti-human ALK-1 antibody(Cat. # MAB370) produced from a hybridoma resulting from the fusion ofmouse myeloma with B cells obtained from a mouse immunized with purifiedNS0-derived recombinant human ALK-1 extracellular domain. We have shownthat this antibody neither neutralizes the interaction between ALK-1 andTGF-beta-1 nor abrogates Smad1 phosphorylation. Rabbit antisera havebeen generated against a synthetic peptide corresponding to a part ofthe intracellular juxtamembrane region of ALK-1 (amino acid residues145-166), coupled to key-hole limpet haemocyanin (KLH) (U.S. Pat. No.6,692,925) and against the entire ALK-1 extracellular domain except forthe leading sequence (Lux et al., J. Biol. Chem., 1999, vol. 274, pp.9984-9992). Abdalla et al (Human Mol. Gen., 2000, vol. 9, pp. 1227-1237)report generation of a polyclonal antibody to ALK-1 using a recombinantvaccinia virus construct. R&D Systems, Inc. makes and sells a polyclonalanti-human ALK-1 antibody (Cat. # AF370) produced in goats immunizedwith purified, NS0-derived, recombinant human ALK-1 extracellulardomain.

To date, no fully human monoclonal antibodies to the ECD of ALK-1 havebeen reported, and no-one has demonstrated the efficacy of anymonoclonal antibody to the ECD of ALK-1 in abrogating theALK-1/TGF-beta-1/Smad1 signaling pathway.

SUMMARY OF THE INVENTION

The invention pertains to isolated neutralizing anti-ALK-1 monoclonalantibodies or antigen-binding portions thereof that bind to primateALK-1, preferably the ECD of primate ALK-1, more preferably the ECD ofhuman ALK-1. In a preferred embodiment, the neutralizing antibodies arefully human monoclonal antibodies or antigen-binding portions thereof.

In another aspect, the present invention is an anti-ALK-1 antibody orantigen-binding portion thereof which antibody or antigen-bindingportion thereof abrogates the ALK-1/TGF-beta-1/Smad1 signaling pathway.In a preferred embodiment, the antibodies are fully human monoclonalantibodies or antigen-binding portions thereof.

In another aspect, the present invention is an anti-ALK-1 antibody orantigen-binding portion thereof which antibody or antigen-bindingportion thereof is an antagonist of TGF-beta-1-stimulated angiogenesis.In a preferred embodiment, the antibodies are fully human monoclonalantibodies or antigen-binding portions thereof.

In another aspect, the present invention is a fully-human anti-ALK-1antibody or antigen-binding portion thereof which antibody orantigen-binding portion thereof is an antagonist ofTGF-beta-1-stimulated tumor angiogenesis.

In another aspect, the present invention is a well-tolerated,injectable, fully-human anti-ALK-1 antibody or antigen-binding portionthereof which antibody or antigen-binding portion thereof is anantagonist of TGF-beta-1-stimulated angiogenesis.

In another aspect, the present invention is an anti-ALK-1 antibody orantigen-binding portion thereof which antibody or antigen-bindingportion thereof inhibits up-regulation of a specific downstream targetgene of ALK-1, Id1. In a preferred embodiment, the antibodies are fullyhuman monoclonal antibodies or antigen-binding portions thereof.

In another aspect, the present invention is an anti-ALK-1 monoclonalantibody or antigen-binding portion thereof wherein the antibody orantigen-binding portion thereof is described in terms of at least one ofseveral functional properties as described below.

For example, in one embodiment the antibody or antigen-binding portionthereof binds to the extracellular domain of primate ALK-1 with anavidity value of 1 μM or less as measured by surface plasmon resonance.In a further embodiment, the antibody or portion binds to theextracellular domain of primate ALK-1 with an avidity value of less than100 nM, less than 5 nM, less than 1 nM, less than 500 pM, less than 100pM, less than 50 pM, less than 20 pM, less than 10 pM, or less than 1pM, as measured by surface plasmon resonance. In certain embodiments,the avidity value is from 0.1 pM to 1 μM. In other embodiments, theavidity value is from 1 pM to 100 nM. In other embodiments, the avidityvalue is from 1 pM to 5 nM. In other embodiments, the avidity value isfrom 1 pM to 500 pM. In other embodiments, the avidity value is from 1pM to 100 pM. In other embodiments, the avidity is from 1 pM to 10 pM.

In another embodiment, the antibody or antigen-binding portion thereofbinds to the extracellular domain of human ALK-1 with an avidity valueof 100 nM or less as measured by surface plasmon resonance. In a furtherembodiment, the antibody or portion binds to the extracellular domain ofhuman ALK-1 with an avidity value of less than 10 nM, less than 5 nM,less than 1 nM, less than 500 pM, less than 100 pM, less than 50 pM,less than 20 pM, less than 10 pM, or less than 1 pM, as measured bysurface plasmon resonance. In certain embodiments, the avidity value isfrom 1 pM to 100 nM. In other embodiments, the avidity value is from 1pM to 5 nM. In other embodiments, the avidity value is from 1 pM to 500pM. In other embodiments, the avidity value is from 1 pM to 100 pM. Inother embodiments, the avidity is from 1 pM to 10 pM.

In another embodiment, the antibody or portion thereof has an off rate(k_(off)) for human ALK-1 of 5 ×10⁻³ s⁻¹ or smaller as measured bysurface plasmon resonance. For example, in certain embodiments theantibody or portion has a k_(off) for human ALK-1 of less than 10⁻³ s⁻¹,less than 5×10⁻⁴ s⁻¹, less than 10⁻⁴ s⁻¹, less than 5×10⁻⁵ s⁻¹, lessthan 10⁻⁵ s⁻¹, or less than 5×10⁻⁶ s⁻¹. In other embodiments, thek_(off) is from 10⁻⁶ s⁻¹ to 10⁻⁴ s⁻¹. In other embodiments, the k_(off)is from 10⁻⁶ s⁻¹ to 5×10⁻⁵ s⁻¹.

In another embodiment, the antibody or portion thereof binds to primateALK-1 with a K_(D) of 1000 nM or less. In a further embodiment, theantibody or portion binds to human ALK-1 with a K_(D) of less than 500nM, less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM,or less than 1 nM, as measured by surface plasmon resonance. In certainembodiments, the K_(D) is from 1 pM to 100 nM. In other embodiments, theK_(D) is from 100 nM to 10 nM. In other embodiments, K_(D) is from 50 nMto 0.1 nM. Such K_(D) values can be measured by any technique knownthose of skill in the art, such as by ELISAs, RIAs, flow cytometry, orsurface plasmon resonance, such as BIACORE®.

In another embodiment, the antibody or portion thereof binds to primateALK-1 with a K_(D) of 1000 nM or less. In a further embodiment, theantibody or portion binds to to human ALK-1 with a K_(D) of less than500 nM, less than 100 nM, less than 50 nM, less than 20 nM, less than 10nM, or less than 1 nM, as measured by surface plasmon resonance. Incertain embodiments, the K_(D) is from 1 μM to 100 nM. In otherembodiments, the K_(D) is from 100 nM to 10 nM. In other embodiments,K_(D) is from 50 nM to 0.1 nM. Such K_(D) values can be measured by anytechnique known those of skill in the art, such as by ELISAs, RIAs, flowcytometry, or surface plasmon resonance, such as BIACORE®.

In another embodiment, the anti-ALK-1 antibody or portion thereof has anIC₅₀ of 500 nM or less as measured by their ability to inhibitup-regulation of a specific downstream target gene of ALK-1, Id1. In afurther embodiment, said IC₅₀ is less than 300 nM, less than 200 nM,less than 150 nM, less than 100 nM, less than 50 nM, less than 20 nM,less than 10 nM, or less than 1 nM. In certain embodiments, the IC₅₀ isfrom 1 nM to 500 nM. In other embodiments, the IC₅₀ is from 5 nM to 250nM. In other embodiments, the IC₅₀ is from 10 nM to 100 nM.

In another embodiment, the anti-ALK-1 antibody or portion thereof has anIC₅₀ of 250 nM or less as measured by their ability to inhibit Smad1phosphorylation determined by Western Blotting using ODYSSEY® InfraredImaging System. In a further embodiment, said IC₅₀ is less than 200 nM,less than 150 nM, less than 100 nM, less than 50 nM, less than 20 nM,less than 10 nM, or less than 1 nM. In certain embodiments, the IC₅₀ isfrom 1 nM to 250 nM. In other embodiments, the IC₅₀ is from 5 nM to 200nM. In other embodiments, the IC₅₀ is from 10 nM to 100 nM.

In another embodiment, the anti-ALK-1 antibody or portion thereofinhibits human vessel angiogenesis in a SCID mouse engrafted with humanforeskin tissue, in which human melanoma M24met tumor cells areintradermally implanted as determined by IHC analysis of human CD-31signal assay by at least 40% as compared to a control sample. In afurther embodiment, the anti-ALK-1 antibody or portion thereof inhibitshuman vessel angiogenesis in a SCID mouse engrafted with human foreskintissue, in which human melanoma M24met tumor cells are intradermallyimplanted by at least 30%, at least 40%, at least 50%, or at least 60%as compared to a control sample.

In another embodiment, the anti-ALK-1 antibody or portion thereof has anEC₅₀ of 500 nM or less as measured by their ability to inhibit humanvessel angiogenesis in a SCID mouse engrafted with human foreskintissue, in which human melanoma M24met tumor cells are intradermallyimplanted. In a further embodiment, said EC₅₀ is less than 400 nM, lessthan 300 nM, less than 200 nM, less than 150 nM, less than 100 nM, lessthan 50 nM, less than 25 nM, or less than 5 nM. In certain embodiments,the EC₅₀ is from 5 nM to 500 nM. In other embodiments, the IC₅₀ is from25 nM to 300 nM. In other embodiments, the IC₅₀ is from 50 nM to 150 nM.

In another embodiment, the anti-ALK-1 antibody or portion thereofinhibits human vessel angiogenesis in a SCID mouse engrafted with humanforeskin tissue, in which a mixture of collagen plus human macrovascularendothelial cells is intradermally implanted as determined by IHCanalysis of human CD-31 signal assay by at least 25% as compared to acontrol sample. In a further embodiment, the anti-ALK-1 antibody orportion thereof inhibits human vessel angiogenesis in a SCID mouseengrafted with human foreskin tissue, in which collagen is intradermallyimplanted by at least 50% as compared to a control sample. In a furtherembodiment, the anti-ALK-1 antibody or portion thereof inhibits by atleast 75%, by at least 80%, by at least 85%, by at least 90% or at least95% as compared to control.

In another embodiment, the anti-ALK-1 antibody or portion thereofcompetes for binding to ALK-1 with an antibody selected from the groupconsisting of 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A);1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1;1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1;4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and5.59.1.

In another embodiment, the anti-ALK-1 antibody or portion thereofcross-competes for binding to ALK-1 with an antibody selected from thegroup consisting of 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A);1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1;1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1;4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and5.59.1.

In another embodiment, the anti-ALK-1 antibody or portion thereof bindsto the same epitope of ALK-1 as an antibody selected from the groupconsisting of 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A);1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1;1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1;4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and5.59.1.

In another embodiment, the anti-ALK-1 antibody or portion thereof bindsto ALK-1 with substantially the same K_(D) as an antibody selected fromthe group consisting of 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A);1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1;1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1;4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and5.59.1.

In another embodiment, the anti-ALK-1 antibody or portion thereof bindsto ALK-1 with substantially the same k_(off) as an antibody selectedfrom the group consisting of 1.11.1; 1.12.1; 1.12.1(rWT);1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1;1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1;4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1;5.57.1; and 5.59.1.

A further aspect of the present invention is an antibody orantigen-binding portion thereof with at least one of the functionalproperties described previously, and comprises a V_(H) domain that is atleast 90% identical in amino acid sequence to any one of SEQ ID NOs: 6;10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78;82; 86; 90; or 104. In one embodiment, said V_(H) domain is at least91%, at least 93%, at least 95%, at least 97%, at least 99%, or 100%identical in amino acid sequence to any one of SEQ ID NOs: 6; 10; 14;18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86;90; or 104.

In a further embodiment, the antibody or portion thereof has at leastone of the functional properties described previously, and comprises aV_(H) domain that is any of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34;38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, ordiffers from any one of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38;42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104 by having atleast one conservative amino acid substitution. For example, the V_(H)domain can differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15conservative amino acid substitutions from any one of SEQ ID NOs: 6; 10;14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82;86; 90; or 104. In a further embodiment, any of these conservative aminoacid substitutions can occur in the CDR1, CDR2, and/or CDR3 regions.

A further aspect of the present invention is an antibody orantigen-binding portion thereof with at least one of the functionalproperties described previously, and comprises a V_(L) domain that is atleast 90% identical in amino acid sequence to any one of SEQ ID NOs: 8;12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80;84; 88; 92; or 127. In one embodiment, said V_(L) domain is at least91%, at least 93%, at least 95%, at least 97%, at least 99%, or 100%identical in amino acid sequence to any one of SEQ ID NOs: 8; 12; 16;20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88;92; or 127.

In a further embodiment, the antibody or portion thereof has at leastone of the functional properties described previously, and comprises aV_(L) domain that is any one of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32;36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, ordiffers from any one of SEQ ID Nos: 8; 12; 16; 20; 24; 28; 32; 36; 40;44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127 by having atleast one conservative amino acid substitution. For example, the V_(L)domain can differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15conservative amino acid substitutions from any one of SEQ ID NOs: 8; 12;16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84;88; 92; or 127. In a further embodiment, any of these conservative aminoacid substitutions can occur in the CDR1, CDR2, and/or CDR3 regions.

Another aspect of the present invention is an antibody orantigen-binding portion thereof with at least one of the functionalproperties described previously wherein the V_(L) and V_(H) domains areeach at least 90% identical in amino acid sequence to the V_(L) andV_(H) domains, respectively, of any one of monoclonal antibodies 1.11.1;1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A);1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1;1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1;5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and 5.59.1. For example, theV_(L) and V_(H) domains are each at least 91%, 93%, 95%, 97%, 99% or100% identical in amino acid sequences to the V_(L) and V_(H) domains,respectively, of any one of monoclonal antibodies 1.11.1; 1.12.1;1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1;1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1;4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1;5.53.1; 5.56.1; 5.57.1; and 5.59.1.

In another aspect of the present invention is a monoclonal antibody orantigen-binding portion thereof that is selected from the groupconsisting of: a) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 6, and a V_(L) domain as set forth inSEQ ID NO: 8; b) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 10, and a V_(L) domain as set forth inSEQ ID NO: 12; c) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 14 and a V_(L) domain as set forth inSEQ ID NO: 16; d) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 18, and a V_(L) domain as set forth inSEQ ID NO: 20; e) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 22 and a V_(L) domain as set forth inSEQ ID NO: 24; f) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 26 and a V_(L) domain as set forth inSEQ ID NO: 28; g) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 30 and a V_(L) domain as set forth inSEQ ID NO: 32; h) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 34 and a V_(L) domain as set forth inSEQ ID NO: 36; i) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 38 and a V_(L) domain as set forth inSEQ ID NO: 40; j) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 42 and a V_(L) domain as set forth inSEQ ID NO: 44; k) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 46 and a V_(L) domain as set forth inSEQ ID NO: 48; 1) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 50 and a V_(L) domain as set forth inSEQ ID NO: 52; m) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 54 and a V_(L) domain as set forth inSEQ ID NO: 56; n) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 58 and a V_(L) domain as set forth inSEQ ID NO: 60; o) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 62 and a V_(L) domain as set forth inSEQ ID NO: 64; p) an antibody or portion thereof that comprises a V_(H)domain as set forth in SEQ ID NO: 66 and a V_(L) domain as set forth inSEQ ID NO: 68; q) an antibody or antigen-binding portion thereof thatcomprises a V_(H) domain as set forth in SEQ ID NO: 70 and a V_(L)domain as set forth in SEQ ID NO: 72; r) an antibody or portion thereofthat comprises a V_(H) domain as set forth in SEQ ID NO: 74 and a V_(L)domain as set forth in SEQ ID NO: 76; s) an antibody or portion thereofthat comprises a V_(H) domain as set forth in SEQ ID NO: 78 and a V_(L)domain as set forth in SEQ ID NO: 80; t) an antibody or portion thereofthat comprises a V_(H) domain as set forth in SEQ ID NO: 82 and a V_(L)domain as set forth in SEQ ID NO: 84; u) an antibody or portion thereofthat comprises a V_(H) domain as set forth in SEQ ID NO: 86 and a V_(L)domain as set forth in SEQ ID NO: 88; v) an antibody or portion thereofthat comprises a V_(H) domain as set forth in SEQ ID NO: 90 and a V_(L)domain as set forth in SEQ ID NO: 92; w) an antibody or portion thereofthat comprises a V_(H) domain as set forth in SEQ ID NO: 104 and a V_(L)domain as set forth in SEQ ID NO: 127; x) an antibody or portion thereofthat comprises a V_(H) domain as set forth in SEQ ID NO: 6 and a V_(L)domain as set forth in SEQ ID NO: 127; and y) an antibody or portionthereof that comprises a V_(H) domain as set forth in SEQ ID NO: 104 anda V_(L) domain as set forth in SEQ ID NO: 8.

In a further embodiment, for any of the antibodies or portions thereofas described above in groups a) to v) the V_(H) and/or V_(L) domains candiffer from the specific SEQ ID NOs recited therein by at least oneconservative amino acid substitution. For example, the V_(H) and/orV_(L) domains can differ from the recited SEQ ID NO by 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutions.In a further embodiment, any of these conservative amino acidsubstitutions can occur in the CDR1, CDR2, and/or CDR3 regions.

In another embodiment, the present invention provides a monoclonalantibody or antigen-binding portion thereof with at least one of thefunctional properties described previously, wherein the V_(H) domain isindependently selected from any one of SEQ ID NOs: 6; 10; 14; 18; 22;26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or104, or a sequence that differs from any one of SEQ ID NOs: 6; 10; 14;18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86;90; or 104, by at least one conservative amino acid substitution, andthe V_(L) domain is independently selected from any one of SEQ ID NOs:8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76;80; 84; 88; 92; or 127, or a sequence that differs from any one of SEQID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68;72; 76; 80; 84; 88; 92; or 127, by at least one conservative amino acidsubstitution. For example, the V_(H) and V_(L) domains can each differfrom SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58;62; 66; 70; 74; 78; 82; 86; 90; or 104, and 8; 12; 16; 20; 24; 28; 32;36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127,respectively, by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15conservative amino acid substitutions.

In a further embodiment, the present invention provides a monoclonalantibody or antigen-binding portion thereof with at least one of thefunctional properties described previously, wherein said antibody orportion comprises V_(H) CDR1, CDR2 and CDR3 sequences independentlyselected from the heavy chain CDR1, CDR2, or CDR3 sequences,respectively, found in any one of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30;34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, or asequence that differs from any one of SEQ ID NOs: 6; 10; 14; 18; 22; 26;30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104,by at least one conservative amino acid substitution. For example, theV_(H) CDR1, CDR2 and CDR3 can differ from the CDR1, CDR2 and CDR3,respectively, of any of SEQ ID NOs: 6; 10; 14; 18; 22; 26; 30; 34; 38;42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90; or 104, by 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acidsubstitutions.

In a further embodiment, the present invention provides a monoclonalantibody or antigen -binding portion thereof with at least one of thefunctional properties described previously, wherein said antibody orportion comprises V_(L) CDR1, CDR2 and CDR3 sequences independentlyselected from the light chain CDR1, CDR2, or CDR3 sequences,respectively, found in any one of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32;36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127, or asequence that differs from any one of SEQ ID NOs: 8; 12; 16; 20; 24; 28;32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127,by at least one conservative amino acid substitution. For example, theV_(L) CDR1, CDR2 and CDR3 can differ from the CDR1, CDR2 and CDR3,respectively, of any of SEQ ID NOs: 8; 12; 16; 20; 24; 28; 32; 36; 40;44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92; or 127 by 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acidsubstitutions.

The present invention further provides a monoclonal antibody orantigen-binding portion thereof with at least one of the functionalproperties described previously, wherein said antibody orantigen-binding portion comprises the V_(H) and V_(L) CDR1, the V_(H)and V_(L) CDR2, and the V_(H) and V_(L) CDR3 as found in any one ofmonoclonal antibodies 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A);1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1;1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1;4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and5.59.1.

The present invention further provides a monoclonal antibody orantigen-binding portion thereof with at least one of the functionalproperties described previously, wherein said antibody orantigen-binding portion comprises a heavy chain that utilizes a humanV_(H) 4-31, V_(H) 3-11, V_(H) 3-15, V_(H) 3-33, V_(H) 4-61 or V_(H) 4-59gene. In some embodiments, the heavy chain utilizes a human V_(H) 3-33gene, a human D 6-19 gene and a human J_(H) 3B gene; a human V_(H) 4-31gene, a human D 6-19 gene and a human J_(H) 4B gene; a human V_(H) 4-61gene, a human D 6-19 gene and a human J_(H) 4B gene; a human V_(H) 4-31gene, a human D 3-3 gene and a human J_(H) 3B gene; a human V_(H) 4-31gene and a human J_(H) 3B gene; a human V_(H) 4-59 gene, a human D 6-19gene and a human J_(H) 4B gene; a human V_(H) 3-11 gene, a human D 3-22gene and a human J_(H) 6B gene; a human V_(H) 3-15 gene, a human D 3-22gene and a human J_(H) 4B gene; a human V_(H) 4-31 gene, a human D 5-12gene and a human J_(H) 6B gene; a human V_(H) 4-31 gene, a human D 4-23gene and a human J_(H) 4B gene; a human V_(H) 4-31 gene, a human D 2-2gene and a human J_(H) 5B gene; a human V_(H) 4-31 gene and a humanJ_(H) 6B gene; human V_(H) 3-15 gene, a human D 1-1 gene and a humanJ_(H) 4B gene; a human V_(H) 3-11 gene, a human D 6-19 gene and a humanJ_(H) 6B gene; a human V_(H) 3-11 gene, a human D 3-10 gene and a humanJ_(H) 6B gene; or a human V_(H) 3-11 gene, a human D 6-6 gene and ahuman J_(H) 6B gene.

The present invention further provides a monoclonal antibody orantigen-binding portion thereof with at least one of the functionalproperties described previously, wherein said antibody orantigen-binding portion comprises a light chain that utilizes a humanV_(κ) A27, V_(κ) A2, V_(κ) A1, V_(κ) A3, V_(κ) B3, V_(κ) B2, V_(κ) L1 orV_(κ) L2 gene. In some embodiments, the light chain utilizes a humanV_(κ) L1 gene and a human J_(κ) 4 gene; a human V_(κ) A27 gene and ahuman J_(κ) 5 gene or a human J_(κ) 4 gene; a human V_(κ) B3 gene and ahuman J_(κ) 1 gene; a human V_(κ) L2 gene and a human J_(κ) 3 gene; ahuman V_(κ) A2 gene and a human J_(κ) 1 gene; a human V_(κ) A3 gene anda human J_(κ) 4 gene; a human V_(κ) A1 gene and a human J_(κ) 1 gene; ahuman V_(κ) B2 gene and a human J_(κ) 4 gene; or a human V_(κ) A2 geneand a human J_(κ) 1 gene.

The present invention further provides a monoclonal antibody orantigen-binding portion thereof with at least one of the functionalproperties described previously, wherein said antibody orantigen-binding portion comprises one or more of a heavy chain and/orlight chain FR1, FR2, FR3 or FR4 amino acid sequence as found in any oneof monoclonal antibodies 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A);1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1;1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1;4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and5.59.1.

The present invention further provides a monoclonal antibody comprisingthe amino acid sequences set forth in: a) SEQ ID NO: 2 and SEQ ID NO: 4;b) SEQ ID NO: 2 and SEQ ID NO: 102; c) SEQ ID NO: 100 and SEQ ID NO: 4;and d) SEQ ID NO: 100 and SEQ ID NO: 102.

In a further embodiment of the present invention is any of theantibodies described previously that is an IgG, an IgM, an IgE, an IgA,or an IgD molecule, or is derived therefrom. For example, the antibodycan be an IgG₁ or IgG₂.

Another embodiment provides any of the antibodies or antigen-bindingportions described above which is an Fab fragment, an F(ab′)₂ fragment,an F_(V) fragment, a single chain Fv fragment, a single chain V_(H)fragment, a single chain V_(L) fragment, a humanized antibody, achimeric antibody or a bispecific antibody.

In a further embodiment is a derivatized antibody or antigen-bindingportion comprising any of the antibodies or portions thereof asdescribed previously and at least one additional molecular entity. Forexample, the at least one additional molecular entity can be a anotherantibody (e.g., a bispecific antibody or a diabody), a detection agent,a label, a cytotoxic agent, a pharmaceutical agent, and/or a protein orpeptide that can mediate association of the antibody or antibody portionwith another molecule (such as a streptavidin core region or apolyhistidine tag). For example, useful detection agents with which anantibody or antigen-binding portion of the invention may be derivatizedinclude fluorescent compounds, including fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin, lanthanide phosphors, and the like. An antibodycan also be labeled with enzymes that are useful for detection, such ashorseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase, glucose oxidase, and the like. In a further embodiment theantibodies or portions thereof of the present invention can also belabeled with biotin, or with a predetermined polypeptide epitoperecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In a still further embodiment of the present invention, any ofthe antibodies or portions thereof can also be derivatized with achemical group such as polyethylene glycol (PEG), a methyl or ethylgroup, or a carbohydrate group.

In some embodiments, the anti-ALK-1 antibodies or antigen bindingportions disclosed herein are attached to a solid support.

In some embodiments, the C-terminal lysine of the heavy chain of any ofthe anti-ALK-1 antibodies of the invention is cleaved. In variousembodiments of the invention, the heavy and light chains of theanti-ALK-1 antibodies may optionally include a signal sequence.

The present invention also provides a pharmaceutical compositioncomprising any of the antibodies or antigen-binding portions thereof asdescribed above and a pharmaceutically acceptable carrier.

In another embodiment, the invention relates to an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes any of theantibodies or antigen binding portions thereof as described herein. Inone particular embodiment, an isolated nucleic acid molecule comprisesthe nucleotide sequence set forth in SEQ ID NO: 1, which sequenceencodes a heavy chain. In another particular embodiment, an isolatednucleic acid molecule comprises the nucleotide sequence set forth in SEQID NO: 3, which sequence encodes a light chain.

In another particular embodiment an isolated nucleic acid moleculecomprises a polynucleotide comprising an open reading frame of the cDNAsequence of a clone deposited under an ATCC accession number PTA-6864.In another particular embodiment an isolated nucleic acid moleculecomprises a polynucleotide comprising an open reading frame of the cDNAsequence of a clone deposited under an ATCC accession number PTA-6865.

In another particular embodiment, an isolated nucleic acid moleculecomprises the nucleotide sequence set forth in SEQ ID NO: 95 or 128,each of which sequences encodes a heavy chain. In another particularembodiment, an isolated nucleic acid molecule comprises the nucleotidesequence set forth in SEQ ID NO: 101, which sequence encodes a lightchain.

The invention further relates to a vector comprising any of the nucleicacid molecules described herein, wherein the vector optionally comprisesan expression control sequence operably linked to the nucleic acidmolecule.

Another embodiment provides a host cell comprising any of the vectorsdescribed herein or comprising any of the nucleic acid moleculesdescribed herein. The present invention also provides an isolated cellline that produces any of the antibodies or antigen-binding portions asdescribed herein or that produces the heavy chain or light chain of anyof said antibodies or said antigen-binding portions.

In another embodiment, the present invention relates to a method forproducing an anti-ALK-1 antibody or antigen-binding portion thereof,comprising culturing any of the host cells or cell lines describedherein under suitable conditions and recovering said antibody orantigen-binding portion.

The present invention also relates to a non-human transgenic animal ortransgenic plant comprising any of the nucleic acids described herein,wherein the non-human transgenic animal or transgenic plant expressessaid nucleic acid.

The present invention further provides a method for isolating anantibody or antigen-binding portion thereof that binds to ALK-1,comprising the step of isolating the antibody from the non-humantransgenic animal or transgenic plant as described herein.

In another embodiment, the invention relates to a hybridoma depositedunder an ATCC accession number of PTA-6808.

The present invention also provides a method for determining if asubstance inhibits up-regulation of a specific downstream target gene ofALK-1, Id1, the method comprising contacting a first sample of cellsthat express Id1 with the substance and determining if Id1 expression isinhibited, wherein a reduced level of Id1 expression in the first sampleof cells contacted with the substance as compared to a control sample ofcells is indicative of said substance inhibiting Id1 expression. Thepresent invention further provides the method, wherein the substance isan antibody that binds to the extracellular domain of ALK-1.

The present invention also provides a method for treating abnormal cellgrowth in a mammal in need thereof, comprising the step of administeringto said mammal any of the antibodies or antigen-binding portionsthereof, or any of the pharmaceutical compositions, as described herein.The present invention further provides a method for treating abnormalcell growth in a mammal in need thereof with an antibody orantigen-binding portion thereof that binds to ALK-1 comprising the stepsof administering to said mammal an effective amount of any of thenucleic acid molecules described herein under suitable conditions thatallow expression of said nucleic acid molecules. In another embodiment,the method of treating abnormal cell growth further comprisesadministering an amount of one or more substances selected fromanti-tumor agents, anti-angiogenesis agents, signal transductioninhibitors, and antiproliferative agents, which amounts are togethereffective in treating said abnormal cell growth. In particularembodiments, said abnormal cell growth is cancerous.

The present invention also provides an isolated Cynomolgus monkey ALK-1protein having an amino acid sequence of SEQ ID NO: 93. The presentinvention further provides an isolated nucleic acid molecule encoding aprotein having an amino acid sequence of SEQ ID NO: 93. The presentinvention further provides an isolated nucleic acid molecule of SEQ IDNO: 94.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of epitope binding data. The 1.12.1(M29I/D19A)antibody was injected for 10 minutes followed by a second 10 minuteinjection of the 1.12.1(M29I/D19A) antibody. This defines the maximumresponse for a 20 minute injection of that antibody. The 20 minuteinjection maximum response was similarly determined for the 1.27.1antibody. The 1.12.1(M29I/D19A) antibody was injected for 10 minutesfollowed by a 10 minute injection of the 1.27.1 antibody. If the totalresponse falls between the defined maximum responses then the twoantibodies must bind to the same epitope. If the total response exceedsthe highest maximum response then the antibodies must bind to differentepitopes. The experiment was repeated with the order of injectionsreversed as described in Example 9.

FIG. 2 shows sequence alignment of human and Cyno ALK-1 proteins.

FIG. 3 shows K_(D) determination of the recombinant 1.12.1 antibodybinding to cell surface ALK-1. (a) Human. (b) Cyno.

1.12.1 (rWT) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb.

1.12.1(M29I/D19A) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing two specific amino acid mutations (methionineat position 29 in the heavy chain replaced with isoleucine and asparticacid at position 19 in the light chain replaced with alanine).

1.12.1(M29I) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing a specific single amino acid mutation wherethe methionine at position 29 in the heavy chain was replaced withisoleucine.

1.12.1(D19A) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing a specific single amino acid mutation wherethe aspartic acid at position 19 in the light chain was replaced withalanine.

FIG. 4 shows examples of ID1 titrations using ID1 TAQMAN® Assay for the1.12.1 antibody variants.

1.12.1 refers to the mAb 1.12.1 variant that was isolated from thehybridoma.

1.12.1(rWT) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb.

1.12.1(M29I/D19A) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing two specific amino acid mutations (methionineat position 29 in the heavy chain replaced with isoleucine and asparticacid at position 19 in the light chain replaced with alanine).

1.12.1(M29I) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing a specific single amino acid mutation wherethe methionine at position 29 in the heavy chain was replaced withisoleucine.

1.12.1(D19A) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing a specific single amino acid mutation wherethe aspartic acid at position 19 in the light chain was replaced withalanine.

FIG. 5 shows examples of ID1 titrations using ID1 TAQMAN® Assay for the1.12.1 antibody sequence variants and the Fab derivative .

1.12.1 refers to the mAb 1.12.1 variant that was isolated from thehybridoma.

1.12.1(rWT) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb.

1.12.1(M29I) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing a specific single amino acid mutation wherethe methionine at position 29 in the heavy chain was replaced withisoleucine.

1.12.1(D19A) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing a specific single amino acid mutation wherethe aspartic acid at position 19 in the light chain was replaced withalanine.

1.12.1(M29I/D19A) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing two specific amino acid mutations (methionineat position 29 in the heavy chain replaced with isoleucine and asparticacid at position 19 in the light chain replaced with alanine).

Fab 1.12.1(M29I/D19A) refers to the Fab fragment of mAb1.12.1(M29I/D19A) prepared by digesting 1.12.1(M29I/D19A) IgG1 usingpapain.

FIG. 6 shows ALK-1 internalization. (a) Monitor neutralizing antibodyremaining on cell surface. (b) Monitor remaining cell surface receptorALK-1.

FIG. 7A shows alignment of variable domain sequences for anti-ALK-1antibodies of the invention, to germline sequences. Mutations comparedto germline are in bold. CDR sequences are underlined. FIG. 7B showsalignment of the predicted amino acid sequences of light chain variabledomains for anti-ALK-1 antibodies 1.12.1, 1.14.1, 1.162.1, 1.31.1,4.62.1 and 4.72.1 to the human germline A27 Vκ sequence. FIGS. 7C and 7Dshow alignment of the predicted amino acid sequences of heavy lightchain variable domains for anti-ALK-1 antibodies 1.12.1, 1.151.1,1.162.1, 1.8.1, 4.24.1, 4.38.1, 4.58.1, 4.62.1, 4.68.1, 4.72.1, 5.13.1and 5.34.1 to the human germline 4-31 V_(H) sequence.

FIG. 8 shows an example of the histological (H & E Staining) analysis ofa section of the engrafted human skin post surgery.

FIG. 9 (A) shows the trichrome staining of collagen in a human skinchimera mouse.

FIG. 9 (B) shows detecting human vessels in the collagen gel implantedin a human foreskin chimera mouse. Tex-red: human vessels. FITC: mousevessels. Yellow: co-staining.

FIG. 10 shows an immunofluorescent image of human (red) and mouse(green) vessels of the M24met tumor in the human foreskin SCID chimeramouse.

FIG. 11 shows the IHC image of human vessels (brown) of the M24met tumorin the human foreskin SCID chimera mouse.

FIG. 12 shows the representative immunofluorescent images of human (red)and mouse (green) vessels of the control and the 1.12.1(M29I/D19A)antibody treated (10 mg/kg) M24met tumors in the human foreskin SCIDchimera mouse.

FIG. 13 shows dose-dependent inhibition of human tumor vessel growth bythe 1.12.1(M29I/D19A) antibody in the human foreskin SCID chimera mousemodel.

FIG. 14 shows the SCID mouse plasma concentration of the1.12.1(M29I/D19A) antibody.

FIG. 15 shows the estimated EC₅₀ for the 1.12.1(M29I/D19A) antibody inthe M24met foreskin SCID-chimera model. The control value at 100% wasgiven an artificial serum concentration of 0.1 nM for graphing purposes.This does not alter the apparent EC₅₀.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclature used in connection with, and techniques of, cell and tissueculture, molecular biology, immunology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook J. & Russell D. Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, John & Sons, Inc. (2002); Harlow and Lane UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols inProtein Science, Wiley, John & Sons, Inc. (2003), incorporated herein byreference. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclature used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the term “ALK-1” refers to mammalian activinreceptor-like kinase-1. The term ALK-1 is intended to includerecombinant ALK-1 and recombinant chimeric forms of ALK-1, which can beprepared by standard recombinant expression methods.

As used herein, the acronym “mAb” refers to a monoclonal antibody.

As used herein, an antibody that is referred to by number is amonoclonal antibody (mAb) that is obtained from the hybridoma of thesame number. For example, monoclonal antibody 1.12.1 is obtained fromhybridoma 1.12.1.

1.12.1 refers to the mAb 1.12.1 variant that was isolated from thehybridoma.

1.12.1(rWT) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb.

1.12.1(M29I/D19A) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing two specific amino acid mutations (methionineat position 29 in the heavy chain replaced with isoleucine and asparticacid at position 19 in the light chain replaced with alanine).

1.12.1(M29I) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing a specific single amino acid mutation wherethe methionine at position 29 in the heavy chain was replaced withisoleucine.

1.12.1(D19A) refers to the mAb 1.12.1 variant that was expressedrecombinant mAb containing a specific single amino acid mutation wherethe aspartic acid at position 19 in the light chain was replaced withalanine.

As used herein, “abnormal cell growth”, unless otherwise indicated,refers to cell growth that is independent of normal regulatorymechanisms (e.g., loss of contact inhibition).

As used herein, the term “adjacent” is used to refer to nucleotidesequences which are directly attached to one another, having nointervening nucleotides. By way of example, the pentanucleotide5′-AAAAA-3′ is adjacent to the trinucleotide 5′-TTT-3′ when the two areconnected thus: 5′-AAAAATTT-3′ or 5′-TTTAAAAA-3′, but not when the twoare connected thus: 5′-AAAAACTTT-3′.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, to “alleviate” a disease, disorder or condition meansreducing the severity of the symptoms of the disease, disorder, orcondition. This includes, but is not limited to, affecting the size,growth and/or mass of a tumor, the extent or progression of metastasis,and the like, in a patient compared with these same parameters in thepatient prior to or in the absence of the method of treatment.

As used herein, the acronym “Id1” refers to a specific downstream targetgene of ALK-1, the Id1 gene, which is important for angiogenesis. TheId1 gene has been reported to control the angiogenesis pathway incertain cancers by turning off the production of a protein,thrombospondin-1 (TSP-1), a naturally occurring angiogenesis suppressor.For example, it has been reported that the Id1 gene, which is highlyexpressed in melanoma, breast, head and neck, brain, cervical, prostate,pancreatic and testicular cancers, results in decreased expression ofTSP-1 and increased tumor blood vessel formation. Volpert, Olga V. etal, “Id1 regulates angiogenesis through transcriptional repression ofthrombospondin-1,” Cancer Cell, December 2002, Vol. 2, pp. 473-483.

As used herein, the term “Smad” refers to Smad domain proteins found ina range of species from nematodes to humans. These highly conservedproteins contain an N-terminal MH1 domain that contacts DNA, and isseparated by a short linker region from the C-terminal MH2 domain, thelatter showing a striking similarity to forkhead-associated (FHA)domains. FHA and Smad (MH2) domains share a common structure consistingof a sandwich of eleven beta strands in two sheets with Greek keytopology. Smad proteins mediate signalling by theTGF-beta/activin/BMP-2/4 cytokines from receptor Ser/Thr protein kinasesat the cell surface to the nucleus. Smad proteins fall into threefunctional classes: the receptor-regulated Smads (R-Smads), includingSmad1, -2, -3, -5, and -8, each of which is involved in aligand-specific signalling pathway; the comediator Smads (co-Smads),including Smad4, which interact with R-Smads to participate insignalling; and the inhibitory Smads (1-Smads), including Smad-6 and -7,which block the activation of R-Smads and Co-Smads, thereby negativelyregulating signalling pathways.

As used herein, the term “TGF-beta” refers to the transforming growthfactors-beta, which constitutes a family of multi-functional cytokines(TGF-beta 1-5) that regulate cell growth and differentiation.Transforming growth factor (TGF) is one of many characterized growthfactors that exist in nature. It plays crucial roles in “SCID” mice withsevere combined immunodeficiency. Many cells synthesize TGF-beta, andessentially all have specific receptors for this peptide. TGF-betaregulates the actions of many other peptide growth factors anddetermines a positive or negative direction of their effects. TGF-betais a tumor suppressing cytokine with growth inhibitory effects inepithelial cells. TGF-β may also act as a tumor promoter by eliciting anepithelial-to-mesenchymal transition. TGF-β inactivates several proteinsinvolved in cell cycle progression and thereby exerts itsgrowth-inhibitory effects on epithelial cells by causing them to arrestin the G1 phase of the cell cycle. The protein functions as adisulphide-linked homodimer. Its sequence is characterised by thepresence of several C-terminal cysteine residues, which forminterlocking disulphide links arranged in a knot-like topology. Asimilar “cystine-knot” arrangement has been noted in the structures ofsome enzyme inhibitors and neurotoxins that bind to voltage-gated Ca²⁺channels, although the precise topology differs. TGF-beta genes areexpressed differentially, suggesting that the various TGF-beta speciesmay have distinct physiological roles in vivo.

As used herein, the term “TGF-beta 1” refers to transforming growthfactor beta receptor type 1, which is a peptide of 112 amino acidresidues derived by proteolytic cleavage from the C-terminal of aprecursor protein. Examination of TGF-beta 1 mRNA levels in adult murinetissues indicates that expression is predominant in spleen, lung andplacenta. TGF-beta 1 is believed to play important roles in pathologicprocesses.

As used herein, the term “SCID” refers to mice with severe combinedimmunodeficiency.

As used herein, the term “HUVEC” refers to human umbilical veinendothelial cells.

As used herein, “amino acids” are represented by the full name thereof,by the three letter code corresponding thereto, or by the one-lettercode corresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.

A “conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chainR group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson, MethodsMol. Biol. 243:307-31 (1994).

Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartic acid and glutamic acid; and 7)sulfur-containing side chains: cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992), herein incorporated by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

In certain embodiments, amino acid substitutions to an anti-ALK-1antibody or antigen-binding portion thereof are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, and (4) conferor modify other physicochemical or functional properties of suchanalogs, but still retain specific binding to ALK-1. Analogs can includevarious substitutions to the normally-occurring peptide sequence. Forexample, single or multiple amino acid substitutions, preferablyconservative amino acid substitutions, may be made in thenormally-occurring sequence, for example in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aminoacid substitutions can also be made in the domain(s) that formintermolecular contacts that can improve the activity of thepolypeptide. A conservative amino acid substitution should notsubstantially change the structural characteristics of the parentsequence; e.g., a replacement amino acid should not alter theanti-parallel β-sheet that makes up the immunoglobulin binding domainthat occurs in the parent sequence, or disrupt other types of secondarystructure that characterizes the parent sequence. In general, glycineand proline would not be used in an anti-parallel β-sheet. Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed., W.H. Freeman and Company, New York (1984)); Introduction to ProteinStructure (C. Branden and J. Tooze, eds., Garland Publishing, New York,N.Y. (1991)); and Thornton et al., Nature 354:105 (1991), incorporatedherein by reference.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “BESTFIT®” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000)). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially blastp or tblastn, using default parameters. See, e.g.,Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul et al.,Nucleic Acids Res. 25:3389-402 (1997); herein incorporated by reference.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.The term “analog” as used herein refers to polypeptides which arecomprised of a segment of at least 25 amino acids that has substantialidentity to a portion of a deduced naturally-occurring amino acidsequence and which has at least one of the properties of thenaturally-occurring polypeptide. Typically, polypeptide analogs comprisea conservative amino acid substitution (or addition or deletion) withrespect to the naturally-occurring sequence. Analogs typically are atleast 20 amino acids long, preferably at least 50 amino acids long orlonger, and can often be as long as a full-length naturally-occurringpolypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987), which are incorporated herein by reference.Such compounds are often developed with the aid of computerizedmolecular modeling.

Peptide mimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a biochemicalproperty or pharmacological activity), such as human antibody, but haveone or more peptide linkages optionally replaced by a linkage selectedfrom the group consisting of: —CH₂NH—, —CH₂S—, —CH₂CH═, —CH═CH— (cis andtrans), —COCH₂—, —CH(OH)CH₂—, and CH₂SO—, by methods well known in theart. Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. In addition,constrained peptides comprising a consensus sequence or a substantiallyidentical consensus sequence variation may be generated by methods knownin the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992),incorporated herein by reference); for example, by adding internalcysteine residues capable of forming intramolecular disulfide bridgeswhich cyclize the peptide.

An intact “antibody” or “immunoglobulin” (Ig) comprises at least twoheavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25kDa) inter-connected by disulfide bonds. There are only two types oflight chain: λ and κ. In humans they are similar, but only one type ispresent in each antibody. Heavy chains are classified as mu, delta,gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD,IgG, IgA, and IgE, respectively. See generally, Fundamental ImmunologyCh. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated byreference in its entirety for all purposes). In a preferred embodiment,the antibody is an IgG and is an IgG1, IgG2, IgG3 or IgG4 subtype. In amore preferred embodiment, the anti-ALK-1 antibody is subclass IgG2.

Each heavy chain is comprised of a heavy chain variable domain (V_(H))and a heavy chain constant region (C_(H)). The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable domain (V_(L)) and a light chainconstant region. The light chain constant region is comprised of onedomain, C_(L). Within light and heavy chains, the variable and constantregions are joined by a “J” region of about 12 or more amino acids, withthe heavy chain also including a “D” region of about 3 or more aminoacids. The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed “complementarity determiningregions” (CDR), interspersed with regions that are more conserved,termed “framework regions” (FR). Each V_(H) and V_(L) is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat, Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987 and 1991)),or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al.,Nature 342:878-883 (1989).

The variable domains of each heavy/light chain pair (V_(H) and V_(L))form the antibody binding site that interacts with an antigen. Thus, anintact IgG antibody, for example, has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The constant regions of the antibodies may mediate the binding ofthe immunoglobulin to host tissues or factors, including various cellsof the immune system (e.g., effector cells) and the first component(Clq) of the classical complement system.

Antibodies must have enough antigen-binding diversity to recognize everypossible pathogen (many V regions) while maintaining the biologicaleffectiveness of their C regions (few C regions). Ig genes are randomlyspliced together from gene segments that allow many V regions to be usedwith a few C regions. Gene segments encoding Ig H, kappa and lambdachains are found on three different chromosomes. During B celldevelopment, recombinase enzymes remove introns and some exons from theDNA and splice segments into functional Ig genes.

Ig gene segments in mammals are arranged in groups of “variable” (V),“diversity” (D), “joining” (J), and “constant” (C) exons. V kappa (Vκ)segments each encode the first two CDR and three FR of the kappa chain Vregion, plus a few residues of CDR3. J kappa (Jκ) segments each encodethe remainder of CDR3 and the fourth FR. C kappa (Cκ) encodes thecomplete C region of the kappa light chain. DNA encoding human kappachain includes approximately 40 functional V kappa (Vκ) segments, five Jkappa (Jκ) segments, and one C kappa (Cλ) gene segment, as well as somegene segments which contain stop codons (“pseudogenes”). Human lambda(λ) chain DNA contains approximately 30 functional V lambda (Vλ)segments and four functional sets of J lambda (Jλ) and C lambda (Cλ)segments. A particular J lambda (Jλ) always pairs with its correspondingC lambda (Cλ), unlike J kappa (Jκ) which all pair with the same C kappa(Cκ). DNA for human H chain includes approximately 50 functional V_(H)segments, 30 D_(H) segments, and six J_(H) segments. The first two CDRand three FR of the heavy chain variable domain are encoded by V_(H).CDR3 is encoded by a few nucleotides of V_(H), all of D_(H), and part ofJ_(H), while FR4 is encoded by the remainder of the J_(H) gene segment.There are also individual gene segments in the DNA for each heavy chaindomain and membrane region of each isotype, arranged in the order inwhich they are expressed by B cells.

The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein”, “isolated polypeptide” or “isolatedantibody” is a protein, polypeptide or antibody that by virtue of itsorigin or source of derivation (1) is not associated with naturallyassociated components that accompany it in its native state, (2) is freeof other proteins from the same species, (3) is expressed by a cell froma different species, or (4) does not occur in nature. Thus, apolypeptide that is chemically synthesized or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A protein mayalso be rendered substantially free of naturally associated componentsby isolation, using protein purification techniques well known in theart.

Examples of isolated antibodies include, but not limited to, ananti-ALK-1 antibody that has been affinity purified using ALK-1, and ananti-ALK-1 antibody that has been synthesized by a cell line in vitro.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein can typically comprise about 50%, 60%, 70%, 80% or 90% w/w ofa protein sample, more usually about 95%, and preferably can be over 99%pure. Protein purity or homogeneity may be indicated by a number ofmeans well known in the art, such as polyacrylamide gel electrophoresisof a protein sample, followed by visualizing a single polypeptide bandupon staining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art of purification.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence. In some embodiments,fragments are at least 5, 6, 8 or 10 amino acids long. In otherembodiments, the fragments are at least 14, at least 20, at least 50, orat least 70, 80, 90, 100, 150 or 200 amino acids long.

The term “analog” or “polypeptide analog” as used herein refers to apolypeptide that comprises a segment that has substantial identity tosome reference amino acid sequence and has substantially the samefunction or activity as the reference amino acid sequence. Typically,polypeptide analogs comprise a conservative amino acid substitution (orinsertion or deletion) with respect to the reference sequence. Analogscan be at least 20 or 25 amino acids long, or can be at least 50, 60,70, 80, 90, 100, 150 or 200 amino acids long or longer, and can often beas long as the full-length polypeptide. Some embodiments of theinvention include polypeptide fragments or polypeptide analog antibodieswith 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17substitutions from the germline amino acid sequence. Fragments oranalogs of antibodies or immunoglobulin molecules can be readilyprepared by those of ordinary skill in the art following the teachingsof this specification.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., ALK-1 or ECD of ALK-1). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H)1 domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546), which consists of a V_(H) domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, V_(L) and V_(H), are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe V_(L) and V_(H) regions pair to form monovalent molecules (known assingle chain Fv (scFv)); see e.g., Bird et al. Science 242:423-426(1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883(1988)). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in whichV_(H) and V_(L) domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448(1993); Poljak et al. Structure 2:1121-1123 (1994)).

Still further, an antibody or antigen-binding portion thereof may bepart of larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov et al. Human Antibodies andHybridomas 6:93-101 (1995)) and use of a cysteine residue, a markerpeptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov et al. Mol. Immunol.31:1047-1058 (1994)). Other examples include where one or more CDRs froman antibody are incorporated into a molecule either covalently ornoncovalently to make it an immunoadhesin that specifically binds to anantigen of interest, such as ALK-1 or ECD of ALK-1. In such embodiments,the CDR(s) may be incorporated as part of a larger polypeptide chain,may be covalently linked to another polypeptide chain, or may beincorporated noncovalently.

Antibody portions, such as Fab and F(ab′)₂ fragments, can be preparedfrom whole antibodies using conventional techniques, such as papain orpepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.

As used herein, the term “human antibody” means any antibody in whichthe variable and constant domain sequences are human sequences. The termencompasses antibodies with sequences derived from human genes, butwhich have been changed, e.g. to decrease possible immunogenicity,increase affinity, eliminate cysteines that might cause undesirablefolding, etc. The term also encompasses such antibodies producedrecombinantly in non-human cells, which might impart glycosylation nottypical of human cells. These antibodies may be prepared in a variety ofways, as described below.

As used herein, the term “neutralizing antibody,” “an inhibitoryantibody” or antagonist antibody means an antibody that inhibits theALK-1/TGF-beta-1/Smad1 signaling pathway. In a preferred embodiment, theantibody inhibits the ALK-1/TGF -beta-1/Smad1 signaling pathway by atleast about 20%, preferably 40%, more preferably 60%, even morepreferably 80%, or even more preferably 85%. Neutralizing or inhibitingpotential of human anti-ALK-1 antibodies may be determined, for example,by their ability to inhibit up-regulation of a specific downstreamtarget gene of ALK-1, Id1, as presented in Example 12; to inhibit Smad1phosphorylation determined by Western Blotting using ODYSSEY® InfraredImaging System from LI-COR Biosciences as presented in Example 13.

The term “chimeric antibody” as used herein means an antibody thatcomprises regions from two or more different antibodies. For example,one or more of the CDRs of a chimeric antibody can be derived from ahuman anti-ALK-1 antibody. In another example, all of the CDRs can bederived from human anti-ALK-1 antibodies. In another example, the CDRsfrom more than one human anti-ALK-1 antibody can be combined in achimeric antibody. For instance, a chimeric antibody may comprise a CDR1from the light chain of a first human anti-ALK-1 antibody, a CDR2 fromthe light chain of a second human anti-ALK-1 antibody and a CDR3 fromthe light chain of a third human anti-ALK-1 antibody, and CDRs from theheavy chain may be derived from one or more other anti-ALK-1 antibodies.Further, the framework regions may be derived from one of the anti-ALK-1antibodies from which one or more of the CDRs are taken or from one ormore different human antibodies. Moreover, as discussed previouslyherein, chimeric antibody includes an antibody comprising a portionderived from the germline sequences of more than one species.

In some embodiments, a chimeric antibody of the invention is a humanizedanti-ALK-1 antibody. A humanized anti-ALK-1 antibody of the inventioncomprises the amino acid sequence of one or more framework regionsand/or the amino acid sequence from at least a portion of the constantregion of one or more human anti-ALK-1 antibodies of the invention andfurther comprises sequences derived from a non-human anti-ALK-1antibody, for example CDR sequences.

As used herein, the term “ELISA” refers to an enzyme-linkedimmunosorbent assay. This assay is well known to those of skill in theart. Examples of this assay can be found in Vaughan, T. J. et al.,Nature Biotech. 14:309-314 (1996), as well as in Example 2 of thepresent application.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE® system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson et al., Ann. Biol. Clin. 51:19-26(1993); Jonsson et al., Biotechniques 11:620-627 (1991); Jonsson et al.,J. Mol. Recognit. 8:125-131 (1995); and Johnsson et al., Anal. Biochem.198:268-277 (1991).

The term “affinity” refers to a measure of the attraction between anantigen and an antibody. The intrinsic attractiveness of the antibodyfor the antigen is typically expressed as the binding affinityequilibrium constant (K_(D)) of a particular antibody-antigeninteraction. An antibody is said to specifically bind an antigen whenthe K_(D) is ≦1 mM, preferably ≦100 nM. A K_(D) binding affinityconstant can be measured by surface plasmon resonance, for example usingthe BIACORE® system as discussed in Examples 7 and 8.

The term “k_(off)” refers to the dissociation rate constant of aparticular antibody-antigen interaction. A k_(off) dissociation rateconstant can be measured by surface plasmon resonance, for example usingthe BIAcore system as discussed in Examples 7 and 8.

The term “avidity” refers to the functional combining strength of anantibody with its antigen which is based on both affinity and valencesof the antibody. As used herein, this term describes the increasedaffinity that occurs as result of multiple antigen binding sites on animmunoglobulin.

As used herein, the term “molecular selectivity” refers to the bindingaffinity of an antibody for a specific antigen being greater than forother antigens. For example, the antibodies of the present invention canbe selective for ALK-1 over ALK-2 through ALK-7, meaning that thebinding affinity of the antibody for ALK-1 is at least 2-fold greater,for example 4-fold, or 10-fold, or 50-fold, or 100-fold or more, thanfor ALK-2 through ALK-7. Such binding affinities can be measured usingstandard techniques known to those of skill in the art.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor or otherwise interactingwith a molecule. Epitopic determinants generally consist of chemicallyactive surface groupings of molecules such as amino acids orcarbohydrate or sugar side chains and generally have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An epitope may be “linear” or “conformational.” In alinear epitope, all of the points of interaction between the protein andthe interacting molecule (such as an antibody) occur linearally alongthe primary amino acid sequence of the protein. In a conformationalepitope, the points of interaction occur across amino acid residues onthe protein that are separated from one another. Once a desired epitopeon an antigen is determined, it is possible to generate antibodies tothat epitope, e.g., using the techniques described in the presentinvention. Alternatively, during the discovery process, the generationand characterization of antibodies may elucidate information aboutdesirable epitopes. From this information, it is then possible tocompetitively screen antibodies for binding to the same epitope. Anapproach to achieve this is to conduct cross-competition studies to findantibodies that competitively bind with one another, i.e. the antibodiescompete for binding to the antigen. A high throughput process for“binning” antibodies based upon their cross-competition is described inInternational Patent Application No. WO 03/48731.

As used herein, the term “binning” refers to a method to groupantibodies based on their antigen binding characteristics. Theassignment of bins is somewhat arbitrary, depending on how different arethe observed binding patterns for all the antibodies tested. Therefore,bins do not always correlate with epitopes determined by other means andshould not be used to define epitopes.

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen-binding portion thereof, competesfor binding to the antigen with a second antibody, or an antigen-bindingportion thereof, where binding of the first antibody with its cognateepitope is detectably decreased in the presence of the second antibodycompared to the binding of the first antibody in the absence of thesecond antibody. The alternative, where the binding of the secondantibody to its epitope is also detectably decreased in the presence ofthe first antibody, can, but need not be the case. That is, a firstantibody can inhibit the binding of a second antibody to its epitopewithout that second antibody inhibiting the binding of the firstantibody to its respective epitope. However, where each antibodydetectably inhibits the binding of the other antibody with its cognateepitope or ligand, whether to the same, greater, or lesser extent, theantibodies are said to “cross-compete” with each other for binding oftheir respective epitope(s). Both competing and cross-competingantibodies are encompassed by the present invention. Regardless of themechanism by which such competition or cross-competition occurs (e.g.,steric hindrance, conformational change, or binding to a common epitope,or portion thereof, and the like), the skilled artisan would appreciate,based upon the teachings provided herein, that such competing and/orcross-competing antibodies are encompassed and can be useful for themethods disclosed herein.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms.

The term “isolated polynucleotide” as used herein means a polynucleotideof genomic, cDNA, or synthetic origin or some combination thereof, whichby virtue of its origin the “isolated polynucleotide” (1) is notassociated with all or a portion of polynucleotides with which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “naturally occurring nucleotides” as used herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” as used herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. AcidsRes. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984);Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-CancerDrug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); U.S. Pat. No. 5,151,510; Uhlmann andPeyman, Chemical Reviews 90:543 (1990), the disclosures of which arehereby incorporated by reference. An oligonucleotide can include a labelfor detection, if desired.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest.

The term “expression control sequence” as used herein meanspolynucleotide sequences that are necessary to effect the expression andprocessing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “vector”, as used herein, means a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Insome embodiments, the vector is a plasmid, i.e., a circular doublestranded piece of DNA into which additional DNA segments may be ligated.In some embodiments, the vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. In some embodiments,the vectors are capable of autonomous replication in a host cell intowhich they are introduced (e.g., bacterial vectors having a bacterialorigin of replication and episomal mammalian vectors). In otherembodiments, the vectors (e.g., non-episomal mammalian vectors) can beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “expressionvectors”).

The term “recombinant host cell” (or simply “host cell”), as usedherein, means a cell into which a recombinant expression vector has beenintroduced. It should be understood that “recombinant host cell” and“host cell” mean not only the particular subject cell but also theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

As used herein, the term “germline” refers to the nucleotide sequencesand amino acid sequences of the antibody genes and gene segments as theyare passed from parents to offspring via the germ cells. This germlinesequence is distinguished from the nucleotide sequences encodingantibodies in mature B cells which have been altered by recombinationand hypermutation events during the course of B cell maturation. Anantibody that “utilizes” a particular germline has a nucleotide or aminoacid sequence that most closely aligns with that germline nucleotidesequence or with the amino acid sequence that it specifies. Suchantibodies frequently are mutated compared with the germline sequence.

The term “percent sequence identity” in the context of nucleic acidsequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides,usually at least about 18 nucleotides, more usually at least about 24nucleotides, typically at least about 28 nucleotides, more typically atleast about 32 nucleotides, and preferably at least about 36, 48 or morenucleotides. There are a number of different algorithms known in the artwhich can be used to measure nucleotide sequence identity. For instance,polynucleotide sequences can be compared using FASTA, Gap or BESTFIT®,which are programs in Wisconsin Package Version 10.0, Genetics ComputerGroup (GCG), Madison, Wis. FASTA, which includes, e.g., the programsFASTA2 and FASTA3, provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000);Pearson, Methods Enzymol. 266:227-258 (1996);Pearson, J. Mol. Biol. 276:71-84 (1998); incorporated herein byreference). Unless otherwise specified, default parameters for aparticular program or algorithm are used. For instance, percent sequenceidentity between nucleic acid sequences can be determined using FASTAwith its default parameters (a word size of 6 and the NOPAM factor forthe scoring matrix) or using Gap with its default parameters as providedin GCG Version 6.1, incorporated herein by reference.

A reference to a nucleotide sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid having aparticular sequence should be understood to encompass its complementarystrand, with its complementary sequence.

The term “substantial similarity” or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, means that whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 85%, preferably at leastabout 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99%of the nucleotide bases, as measured by any well-known algorithm ofsequence identity, such as FASTA, BLAST or Gap, as discussed above.

The term “percent sequence identity” in the context of amino acidsequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about five amino acids,usually at least about 20 amino acids, more usually at least about 30amino acids, typically at least about 50 amino acids, more typically atleast about 100 amino acids, and even more typically about 150, 200 ormore amino acids. There are a number of different algorithms known inthe art that can be used to measure amino acid sequence identity. Forinstance, amino acid sequences can be compared using FASTA, Gap orBESTFIT®, which are programs in Wisconsin Package Version 10.0, GeneticsComputer Group (GCG), Madison, Wis.

As applied to polypeptides, the term “substantial identity” or“substantial similarity” means that two amino acid sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT® using defaultgap weights as supplied with the programs, share at least 70%, 75% or80% sequence identity, preferably at least 90% or 95% sequence identity,and more preferably at least 97%, 98% or 99% sequence identity. Incertain embodiments, residue positions that are not identical differ byconservative amino acid substitutions.

The term “signal sequence,” also called signal peptide, leader peptide,refers to a segment of about 15 to 30 amino acids at the N terminus of aprotein that enables the protein to be secreted (pass through a cellmembrane). The signal sequence is removed as the protein is secreted.

As used herein, the terms “label” or “labeled” refers to incorporationof another molecule in the antibody. In one embodiment, the label is adetectable marker, e.g., incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). In another embodiment, the label or marker can be therapeutic,e.g., a drug conjugate or toxin. Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y,99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, TAXOL® (paclitaxel), cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. In some embodiments, labelsare attached by spacer arms of various lengths to reduce potentialsteric hindrance.

The term “primate” refers to a mammal of the order primates, whichincludes the anthropoids and prosimians, characterized by refineddevelopment of the hands and feet, a shortened snout, and a large brain.The mammalian order Primates includes humans, apes, monkeys, andprosimians, or lower primates.

“Therapeutically effective amount” refers to that amount of thetherapeutic agent being administered which will relieve to some extentone or more of the symptoms of the disorder being treated. In referenceto the treatment of cancer, a therapeutically effective amount refers tothat amount which has at least one of the following effects: reducingthe size of the tumor; inhibiting (that is, slowing to some extent,preferably stopping) tumor metastasis; inhibiting to some extent (thatis, slowing to some extent, preferably stopping) tumor growth, andrelieving to some extent (or, preferably, eliminating) one or moresymptoms associated with the cancer.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabrogating a biological disorder and/or its attendant symptoms. Withregard to cancer, these terms simply mean that the life expectancy of anindividual affected with a cancer will be increased or that one or moreof the symptoms of the disease will be reduced.

“Contacting” refers to bringing an antibody or antigen binding portionthereof of the present invention and a target ALK-1, or epitope thereof,together in such a manner that the antibody can affect the biologicalactivity of the ALK-1. Such “contacting” can be accomplished “in vitro,”i.e., in a test tube, a petri dish, or the like. In a test tube,contacting may involve only an antibody or antigen binding portionthereof and ALK-1 or epitope thereof or it may involve whole cells.Cells may also be maintained or grown in cell culture dishes andcontacted with antibodies or antigen binding portions thereof in thatenvironment. In this context, the ability of a particular antibody orantigen binding portion thereof to affect a ALK-1-related disorder,i.e., the IC₅₀ of the antibody, can be determined before use of theantibody in vivo with more complex living organisms is attempted. Forcells outside the organism, multiple methods exist, and are well-knownto those skilled in the art, to contact ALK-1 with the antibodies orantigen-binding portions thereof.

The acronym “FACS” refers to Fluorescence Activated Cell Sorting. Theacronym FACS and flow cytometry are used interchangeably. Fluorescentlabeling allows investigation of cell structure and function.Immunofluorescence, the most widely used application, involves thestaining of cells with antibodies conjugated to fluorescent dyes such asfluorescein and phycoerythrin. This method is often used to labelmolecules on the cell surface, but antibodies can be directed at targetsin cytoplasm. In direct immunofluorescence an antibody to a molecule isdirectly conjugated to a fluorescent dye, and cells are stained in onestep. In indirect immunofluorescence the primary antibody is notlabeled, and a second fluorescently conjugated antibody is added whichis specific for the first antibody.

Anti-ALK-1 Antibodies

This invention pertains to isolated neutralizing anti-ALK-1 monoclonalantibodies or antigen-binding portions thereof that bind to primateALK-1, preferably the ECD of primate ALK-1, more preferably the ECD ofhuman ALK-1. In a preferred embodiment, the invention pertains toisolated neutralizing antibodies that are fully human monoclonalantibodies or antigen-binding portions thereof. Preferably, the humanantibodies are recombinant human anti-ALK-1 antibodies that have greateraffinity for ALK-1 than for ALK-2 through ALK-7. In some embodiments,human anti-ALK-1 antibodies are produced by immunizing a non-humantransgenic animal, e.g., a rodent, whose genome comprises humanimmunoglobulin genes so that the transgenic animal produces humanantibodies. Various aspects of the invention relate to such antibodiesand antigen-binding portions, and pharmaceutical compositions thereof,as well as nucleic acids, recombinant expression vectors and host cellsfor making such antibodies and antigen-binding portions. Methods ofusing the antibodies and antigen-binding portions of the presentinvention to abrogate the ALK-1/TGF-beta-1/Smad1 signaling pathway or todetect ALK-1, either in vitro or in vivo, are also encompassed by theinvention.

An anti-ALK-1 antibody of the invention can comprise a human kappa or ahuman lambda light chain or an amino acid sequence derived therefrom. Insome embodiments comprising a kappa light chain, the light chainvariable domain (V_(L)) utilizes a human A27, A2, A1, A3, B3, B2, L1 orL2 V_(κ) gene. In some embodiments, the light chain utilizes a humanV_(κ) L1 gene and a human J_(κ) 4 gene; a human V_(κ) A27 gene and ahuman J_(κ) 5 gene or a human J_(κ) 4 gene; a human V_(κ) B3 gene and ahuman J_(κ) 1 gene; a human V_(κ) L2 gene and a human J_(κ) 3 gene; ahuman V_(κ) A2 gene and a human J_(κ) 1 gene; a human V_(κ) A3 gene anda human J_(κ) 4 gene; a human V_(κ) A1 gene and a human J_(κ) 1 gene; ahuman V_(κ) B2 gene and a human J_(κ) 4 gene; or a human V_(κ) A2 geneand a human J_(κ) 1 gene.

In some embodiments, the V_(L) of the anti-ALK-1 antibody comprises oneor more amino acid substitutions, deletions or insertions (additions)relative to the germline V_(κ) amino acid sequence. In some embodiments,the V_(L) of the anti-ALK-1 antibody comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 or 15 amino acid substitutions relative to thegermline V_(κ) amino acid sequence. In some embodiments, one or more ofthe substitutions from germline is in the CDR regions of the lightchain. In some embodiments, the V_(κ) amino acid substitutions relativeto germline are at one or more of the same positions as thesubstitutions relative to germline found in any one or more of the V_(L)of the antibodies provided herein as shown, for example, at FIG. 7. Insome embodiments, the amino acid changes are at one or more of the samepositions, but involve a different substitution than in the referenceantibody.

In some embodiments, amino acid substitutions relative to germline occurat one or more of the same positions as substitutions from germline inany of the V_(L) of antibodies 1.11.1; 1.12.1; 1.12.1(rWT); 1.13.1;1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1;4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1;5.53.1; 5.56.1; 5.57.1; and 5.59.1, but the substitutions may representconservative amino acid substitutions at such position(s) relative tothe amino acid in the reference antibody. For example, if a particularposition in one of these antibodies is changed relative to germline andis glutamate, one may substitute aspartate at that position. Similarly,if an amino acid substitution compared to germline in an exemplifiedantibody is serine, one may conservatively substitute threonine forserine at that position. Conservative amino acid substitutions arediscussed supra.

In some embodiments, the anti-ALK-1 antibody comprises a light chainamino acid sequence of SEQ ID NO: 4. In other embodiments, the lightchain comprises the light chain amino acid sequence of antibody 1.11.1;1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A);1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1;1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1;5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1.

In some embodiments, the light chain of the human anti-ALK-1 antibodycomprises the V_(L) amino acid sequence of antibody 1.12.1 (SEQ ID NO:8); 1.11.1 (SEQ ID NO: 12); 1.13.1 (SEQ ID NO: 16); 1.14.1 (SEQ ID NO:20); 1.151.1 (SEQ ID NO: 24); 1.162.1 (SEQ ID NO: 28); 1.183.1 (SEQ IDNO: 32); 1.8.1(SEQ ID NO: 36); 1.9.1(SEQ ID NO: 40); 4.10.1 (SEQ ID NO:44); 4.24.1 (SEQ ID NO: 48); 4.38.1(SEQ ID NO: 52); 4.58.1 (SEQ ID NO:56); 4.62.1 (SEQ ID NO: 60); 4.68.1 (SEQ ID NO: 64); 4.72.1 (SEQ ID NO:68); 5.13.1 (SEQ ID NO: 72); 5.34.1 (SEQ ID NO: 76); 5.53.1 (SEQ ID NO:80); 5.56.1 (SEQ ID NO: 84); 5.57.1 (SEQ ID NO: 88); or 5.59.1 (SEQ IDNO: 92); or said amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutionsand/or a total of up to 3 non-conservative amino acid substitutions. Inother embodiments the light chain of the human anti-ALK-1 antibodycomprises the V_(L) amino acid sequence of antibody 1.27.1; 1.29.1 or1.31.1. In some embodiments, the light chain comprises the amino acidsequence from the beginning of the CDR1 to the end of the CDR3 of anyone of the foregoing antibodies.

In some embodiments, the light chain may comprise the amino acidsequences of CDR1, CDR2 and CDR3 regions independently selected from thelight chain CDR1, CDR2 and CDR3 regions, respectively, of two or moremonoclonal antibodies selected from 1.11.1; 1.12.1; 1.12.1(rWT);1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1;1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1;4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1;5.57.1; or 5.59.1, or said CDR regions each having less than 3 or lessthan 2 conservative amino acid substitutions and/or a total of three orfewer non-conservative amino acid substitutions.

In certain embodiments, the light chain of the anti-ALK-1 antibodycomprises the amino acid sequences of the light chain CDR1, CDR2 andCDR3 regions of an antibody selected from 1.11.1; 1.12.1; 1.12.1(rWT);1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1;1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1;4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1;5.57.1; or 5.59.1, or said CDR regions each having less than 3 or lessthan 2 conservative amino acid substitutions and/or a total of three orfewer non-conservative amino acid substitutions.

With regard to the heavy chain, in some embodiments, the variable domain(V_(H)) utilizes a human V_(H) 4-31, V_(H) 3-11, V_(H) 3-15, V_(H) 3-33,V_(H) 4-61 or V_(H) 4-59 gene. In some embodiments, the V_(H) sequenceof the anti-ALK-1 antibody contains one or more amino acidsubstitutions, deletions or insertions (additions), collectively“mutations”, relative to the germline V_(H) amino acid sequence. In someembodiments, the variable domain of the heavy chain comprises 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or 11 mutations from the germline V_(H) amino acidsequence. In some embodiments, the mutation(s) are non-conservativesubstitutions compared to the germline amino acid sequence. In someembodiments, the mutations are in the CDR regions of the heavy chain. Insome embodiments, the heavy chain utilizes a human V_(H) 3-33 gene, ahuman D 6-19 gene and a human J_(H) 3B gene; a human V_(H) 4-31 gene, ahuman D 6-19 gene and a human J_(H) 4B gene; a human V_(H) 4-61 gene, ahuman D 6-19 gene and a human J_(H) 4B gene; a human V_(H) 4-31 gene, ahuman D 3-3 gene and a human J_(H) 3B gene; a human V_(H) 4-31 gene anda human J_(H) 3B gene; a human V_(H) 4-59 gene, a human D 6-19 gene anda human J_(H) 4B gene; a human V_(H) 3-11 gene, a human D 3-22 gene anda human J_(H) 6B gene; a human V_(H) 3-15 gene, a human D 3-22 gene anda human J_(H) 4B gene; a human V_(H) 4-31 gene, a human D 5-12 gene anda human J_(H) 6B gene; a human V_(H) 4-31 gene, a human D 4-23 gene anda human J_(H) 4B gene; a human V_(H) 4-31 gene, a human D 2-2 gene and ahuman J_(H) 5B gene; a human V_(H) 4-31 gene and a human J_(H) 6B gene;human V_(H) 3-15 gene, a human D 1-1 gene and a human J_(H) 4B gene; ahuman V_(H) 3-11 gene, a human D 6-19 gene and a human J_(H) 6B gene; ahuman V_(H) 3-11 gene, a human D 3-10 gene and a human J_(H) 6B gene; ora human V_(H) 3-11 gene, a human D 6-6 gene and a human J_(H) 6B gene.

In some embodiments, amino acid substitutions are at one or more of thesame positions as the substitutions from germline in any one or more ofthe V_(H) of antibodies 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A);1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1;1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1;4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or5.59.1. In other embodiments, the amino acid changes are at one or moreof the same positions but involve a different substitution than in thereference antibody.

In some embodiments, the heavy chain comprises an amino acid sequence ofSEQ ID NO: 2. In other embodiments, the heavy chain comprises the heavychain amino acid sequence of antibody 1.11.1; 1.12.1; 1.12.1(rWT);1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1;1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1;4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1;5.57.1; or 5.59.1. In some embodiments, the heavy chain comprises theV_(H) amino acid sequence of antibody 1.12.1 (SEQ ID NOS: 6); 1.11.1(SEQ ID NO: 10); 1.13.1 (SEQ ID NO: 14); 1.14.1 (SEQ ID NO: 18); 1.151.1(SEQ ID NO: 22); 1.162.1 (SEQ ID NO: 26); 1.183.1 (SEQ ID NO: 30);1.8.1(SEQ ID NO: 34); 1.9.1(SEQ ID NO: 38); 4.10.1 (SEQ ID NO: 42);4.24.1 (SEQ ID NO: 46); 4.38.1(SEQ ID NO: 50); 4.58.1 (SEQ ID NO: 54);4.62.1 (SEQ ID NO: 58); 4.68.1 (SEQ ID NO: 62); 4.72.1 (SEQ ID NO: 66);5.13.1 (SEQ ID NO: 70); 5.34.1 (SEQ ID NO: 74); 5.53.1 (SEQ ID NO: 78);5.56.1 (SEQ ID NO: 82); 5.57.1 (SEQ ID NO: 86); or 5.59.1 (SEQ ID NO:90); or said V_(H) amino acid sequence having up to 1, 2, 3, 4, 6, 8, 9,10 or 11 conservative amino acid substitutions and/or a total of up to 3non-conservative amino acid substitutions. In other embodiments, theheavy chain comprises the V_(H) amino acid sequence of antibody 1.27.1;1.29.1 or 1.31.1. In some embodiments, the heavy chain comprises theamino acid sequence from the beginning of the CDR1 to the end of theCDR3 of any one of the foregoing antibodies.

In some embodiments, the heavy chain comprises the heavy chain CDR1,CDR2 and CDR3 regions of antibody 1.11.1; 1.12.1; 1.12.1(rWT);1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1;1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1;4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1;5.57.1; or 5.59.1, or said CDR regions each having less than 8, lessthan 6, less than 4, or less than 3 conservative amino acidsubstitutions and/or a total of three or fewer non-conservative aminoacid substitutions.

In some embodiments, the heavy chain CDR regions are independentlyselected from the CDR regions of two or more antibodies selected fromantibodies 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I);1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1;1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1;4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1. In anotherembodiment, the antibody comprises a light chain as disclosed above anda heavy chain as disclosed above. In a further embodiment, the lightchain CDRs and the heavy chain CDRs are from the same antibody.

In various embodiments, the anti-ALK-1 antibodies have the full-lengthheavy chain and full length light chain amino acid sequence(s), theV_(H) and V L amino acid sequences, the heavy chain CDR1, CDR2 and CDR3and light chain CDR1, CDR2 and CDR3 amino acid sequences or the heavychain amino acid sequence from the beginning of the CDR1 to the end ofthe CDR3 and the light chain amino acid sequence from the beginning ofthe CDR1 to the end of the CDR3 of an anti-ALK-1 antibody providedherein.

One type of amino acid substitution that may be made is to change one ormore cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. In oneembodiment, there is a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant domain of an antibody. In some embodiments,the cysteine is canonical.

Another type of amino acid substitution that may be made is to removepotential proteolytic sites in the antibody. Such sites may occur in aCDR or framework region of a variable domain or in the constant domainof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of heterogeneity in the antibodyproduct and thus increase its homogeneity. Another type of amino acidsubstitution is to eliminate asparagine-glycine pairs, which formpotential deamidation sites, by altering one or both of the residues.

In some embodiments, the C-terminal lysine of the heavy chain of theanti ALK-1 antibody of the invention is cleaved. In various embodimentsof the invention, the heavy and light chains of the anti-ALK-1antibodies may optionally include a signal sequence.

In one aspect, the invention provides twenty five inhibitory humananti-ALK-1 monoclonal antibodies and the hybridoma cell lines thatproduce them. In certain embodiments, antibodies of the presentinvention are IgGs designated as: 1.11.1; 1.12.1; 1.12.1(rWT);1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1;1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1;4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1;5.57.1; and 5.59.1. In preferred embodiments, the human anti-ALK-1antibody is antibody 1.12.1, 1.12.1(M29I/D19A), 1.12.1(M29I),1.12.1(D19A), 1.27.1, 1.14.1, 1.162.1, 1.31.1, 4.62.1 or 4.72.1.

Antibodies recognize surface-exposed epitopes on antigens as regions oflinear (primary) sequence or structural (secondary) sequence. BIAcorewas used in order to define the functional epitope landscape anddetermine the epitope exclusivity of the anti-ALK-1 antibodiesexemplified by this invention.

Table 1 lists the sequence identifiers (SEQ ID NO) of the nucleic acidsencoding the full-length heavy and light chains of 1.12.1 antibodyvariants and variable domain-containing portions of anti-ALK-1antibodies of the invention, and the corresponding deduced amino acidsequences.

TABLE 1 SEQUENCE IDENTIFIERS (SEQ ID NO) V DOMAIN FULL LENGTH CONTAININGPORTION Heavy Light Heavy Light Antibody DNA PROTEIN DNA PROTEIN DNAPROTEIN DNA PROTEIN 1.11.1 9 10 11 12 1.12.1(M29I/D19A) 1 2 3 4 5 6 7 81.12.1 95 100 101 102 103 104 126 127 1.12.1(rWT) 128 100 101 102 129104 126 127 1.13.1 13 14 15 16 1.14.1 17 18 19 20 1.151.1 21 22 23 241.162.1 25 26 27 28 1.183.1 29 30 31 32 1.8.1 33 34 35 36 1.9.1 37 38 3940 4.10.1 41 42 43 44 4.24.1 45 46 47 48 4.38.1 49 50 51 52 4.58.1 53 5455 56 4.62.1 57 58 59 60 4.68.1 61 62 63 64 4.72.1 65 66 67 68 5.13.1 6970 71 72 5.34.1 73 74 75 76 5.53.1 77 78 79 80 5.56.1 81 82 83 84 5.57.185 86 87 88 5.59.1 89 90 91 92 1.12.1(M29I/D19A) refers to theanti-ALK-1 antibody containing a specific single amino acid mutation inthe heavy chain where the methionine at position 29 was replaced withisoleucine and a specific single amino acid mutation in the light chainwhere the aspartic acid at position 19 was replaced with alanine asdescribed in Example 4. 1.12.1 refers to the mAb 1.12.1 variant that wasisolated from the hybridoma. 1.12.1(rWT) refers to the mAb 1.12.1variant that was expressed as a recombinant mAb described in Example 3.

The invention further provides heavy and/or light chain variants ofcertain of the above-listed human anti-ALK-1 antibodies, comprising oneor more amino acid modifications. To designate the variants, the firstletter is the one letter symbol for the amino acid of thenaturally-occurring antibody chain, the number refers to the position ofthe amino acid (wherein position one is the N-terminal amino acid of theFR1), and the second letter is the one letter symbol for the variantamino acid.

In still further embodiments, the invention includes antibodiescomprising variable domain amino acid sequences with more than 80%, morethan 85%, more than 90%, more than 95%, more than 96%, more than 97%,more than 98% or more than 99% sequence identity to a variable domainamino acid sequence of any of the above-listed human anti-ALK-1antibodies.

Class and Subclass of Anti-ALK-1 Antibodies

The class and subclass of anti-ALK-1 antibodies may be determined by anymethod known in the art. In general, the class and subclass of anantibody may be determined using antibodies that are specific for aparticular class and subclass of antibody. Such antibodies are availablecommercially. The class and subclass can be determined by ELISA, WesternBlot as well as other techniques. Alternatively, the class and subclassmay be determined by sequencing all or a portion of the constant domainsof the heavy and/or light chains of the antibodies, comparing theiramino acid sequences to the known amino acid sequences of various classand subclasses of immunoglobulins, and determining the class andsubclass of the antibodies.

The class of an anti-ALK-1 antibody obtained as described above may beswitched with another. In one aspect of the invention, a nucleic acidmolecule encoding V_(L) or V_(H) is isolated using methods well-known inthe art such that it does not include nucleic acid sequences encodingC_(L) or C_(H) . “Antibody Engineering” (Kontermann & Dubel, Eds.,Springer-Verlag, Berlin (2001)). The nucleic acid molecules encodingV_(L) or V_(H) are then operatively linked to a nucleic acid sequenceencoding a C_(L) or C_(H), respectively, from a different class ofimmunoglobulin molecule. This may be achieved using a vector or nucleicacid molecule that comprises a C_(L) or C_(H) chain, as described above.For example, an anti-ALK-1 antibody that was originally IgM may be classswitched to an IgG. Further, the class switching may be used to convertone IgG subclass to another, e.g., from IgG1 to IgG2. A preferred methodfor producing an antibody of the invention comprising a desired isotypescomprises the steps of isolating a nucleic acid molecule encoding theheavy chain of an anti-ALK-1 antibody and a nucleic acid moleculeencoding the light chain of an anti-ALK-1 antibody, obtaining thevariable domain of the heavy chain, ligating the variable domain of theheavy chain with the constant domain of a heavy chain of the desiredisotype, expressing the light chain and the ligated heavy chain in acell, and collecting the anti-ALK-1 antibody with the desired isotype.

In some embodiments, the anti-ALK-1 antibody is a monoclonal antibody.The anti-ALK-1 antibody can be an IgG, an IgM, an IgE, an IgA, or an IgDmolecule. In a preferred embodiment, the anti-ALK-1 antibody is an IgGand is an IgG1, IgG2, IgG3, IgG4 subclass. In another preferredembodiment, the antibody is subclass IgG2.

Identification of ALK-1 Epitopes Recognized by Anti-ALK-1 Antibodies

The invention provides a human anti-ALK-1 monoclonal antibody that bindsto ALK-1 and competes or cross-competes with and/or binds the sameepitope as: (a) an antibody selected from 1.11.1; 1.12.1; 1.12.1(rWT);1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1;1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1;4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1;5.57.1; and 5.59.1; (b) an antibody that comprises a heavy chainvariable domain having the amino acid sequence of the V_(H) domain inany one of SEQ ID NOS: 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50;54; 58; 62; 66; 70; 74; 78; 82; 86; 90 or 104, (c) an antibody thatcomprises a light chain variable domain having the amino acid sequenceof the V_(L) domain in any one of SEQ ID NOS: and 8; 12; 16; 20; 24; 28;32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80; 84; 88; 92 or 127,(d) an antibody that comprises both a heavy chain variable domain asdefined in (b) and a light chain variable domain as defined in (c).

One can determine whether an antibody binds to the same epitope or crosscompetes for binding with an anti-ALK-1 antibody by using methods knownin the art. In one embodiment, one allows the anti-ALK-1 antibody of theinvention to bind to ALK-1 under saturating conditions and then measuresthe ability of the test antibody to bind to ALK-1. If the test antibodyis able to bind to ALK-1 at the same time as the reference anti-ALK-1antibody, then the test antibody binds to a different epitope than thereference anti-ALK-1 antibody. However, if the test antibody is not ableto bind to ALK-1 at the same time, then the test antibody binds to thesame epitope, an overlapping epitope, or an epitope that is in closeproximity to the epitope bound by the anti-ALK-1 antibody of theinvention. This experiment can be performed using ELISA, RIA, BIACORE®,or flow cytometry. To test whether an anti-ALK-1 antibody cross-competeswith another anti-ALK-1 antibody, one may use the competition methoddescribed above in two directions, i.e. determining if the knownantibody blocks the test antibody and vice versa. In a preferredembodiment, the experiment is performed using BIACORE®.

Binding Affinity of Anti-ALK-1 Antibodies to ALK-1

The binding affinity (K_(D)) and dissociation rate (k_(off)) of ananti-ALK-1 antibody or antigen-binding portion thereof to ALK-1 can bedetermined by methods known in the art. The binding affinity can bemeasured by ELISAs, RIAs, flow cytometry, or surface plasmon resonance,such as BIACORE®. The dissociation rate can be measured by surfaceplasmon resonance. Preferably, the binding affinity and dissociationrate is measured by surface plasmon resonance. More preferably, thebinding affinity and dissociation rate are measured using BIACORE®. Onecan determine whether an antibody has substantially the same K_(D) as ananti-ALK-1 antibody by using methods known in the art. Such methods ofdetermining K_(D) and k_(off) can be used during the initial screeningstage, as well as during subsequent optimization stages.

Inhibition of ALK-1 Activity by Anti-ALK-1 Antibody

Anti-ALK-1 monoclonal antibodies that inhibit ALK-1 binding can beidentified using a number of assays. For example, neutralizinganti-ALK-1 antibodies can be identified by their inhibition ofup-regulation of a specific downstream target gene of ALK-1, Id1, asdescribed in Example 12. Preferred anti-ALK-1 antibodies have an IC₅₀ ofno more than 500 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10nM, or 1 nM.

One also can determine the ability of an anti-ALK-1 antibody to inhibitSmad1 phosphorylation determined by Western Blotting using ODYSSEY®Infrared Imaging System, as described in Example 13. In variousembodiments, the anti-ALK-1 antibody has an IC₅₀ in this assay of nomore than 250 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 1 nM.

Inhibition of Angiogenesis by Anti-ALK-1 Antibody

In another embodiment, the anti-ALK-1 antibody or portion thereofinhibits human vessel angiogenesis as demonstrated in a SCID mouseengrafted with human foreskin tissue, in which human melanoma M24mettumor cells are intradermally implanted as determined by IHC analysis ofhuman CD-31 signal assay by a factor of at least 40% as compared to acontrol sample as described in Example 17 and shown in Table 13.

In another embodiment, the anti-ALK-1 antibody or portion thereofinhibits human vessel angiogenesis as demonstrated in a SCID mouseengrafted with human foreskin tissue, in which collagen is intradermallyimplanted as determined by IHC analysis of human CD-31 signal assay by afactor of at least 50% as compared to a control sample as described inExample 16 and shown in Table 12.

Species and Molecular Selectivity

In another aspect of the invention, the anti-ALK-1 antibodiesdemonstrate both species and molecular selectivity. Following theteachings of the specification, one may determine the species ormolecular selectivity for the anti-ALK-1 antibody using methods wellknown in the art. For instance, one may determine the speciesselectivity using Western blot, surface plasmon resonance, e.g.,BIAcore, ELISA, immunoprecipitation or RIA.

In some embodiments, the anti-ALK-1 antibody binds to primate ALK-1 witha K_(D) that is at least two times smaller than its K_(D) for rodentALK-1. In a further embodiment, the K_(D) for primate ALK-1 is at least3-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least200-fold, at least 500-fold, or at least 1000-fold smaller than itsK_(D) for rodent ALK-1 as measured by flow cytometry.

In other embodiments, the anti-ALK-1 antibody has a selectivity forALK-1 over ALK-2 through ALK-7. In some embodiments, the anti-ALK-1antibody does not exhibit any appreciable specific binding to any otherprotein other than ALK-1. Preferably, the anti-ALK-1 antibody binds tothe ECD of human ALK-1.

Methods of Producing Antibodies and Antibody Producing Cell Lines

ALK-1 Immunogen

In some embodiments, the ALK-1 immunogen or antigen is isolated and/orpurified ALK-1. In some embodiments, the ALK-1 immunogen is human ALK-1.In preferred embodiments, the ALK-1 immunogen is the ECD of human ALK-1.Human ALK-1, or antigenic portions thereof, can be prepared according tomethods well known to those in the art, or can be purchased fromcommercial vendors. The human ALK-1 amino acid and nucleotide sequencesare known (see e.g. Genbank record Accession No. L17075). ACVRL1 geneencoding a full-length ALK-1 is commercially available from InvitrogenInc., Clone ID IOH21048. For example, R&D Systems, Inc. sells therecombinant human ALK-1/Fc chimera (Catalog Number 370-AL) prepared byexpression of a DNA sequence encoding the ECD amino acid residues 1-118of ALK-1, which DNA sequence was fused to a DNA sequence encoding theF_(c) region of human IgG via a DNA sequence encoding a polypeptidelinker in a mouse myeloma cell line. The recombinant mature humanALK-1/Fc chimera is a disulfide-linked homodimeric protein having Asp 22at the amino-terminus. In addition, Example 1 describes preparation ofALK-1 ECD His-Tag protein which has been used for generation ofhybridomas producing an anti-ALK-1 antibody according to the presentinvention.

In other embodiments, the ALK-1 antigen is a cell that expresses oroverexpresses ALK-1. In other embodiments, the ALK-1 antigen is arecombinant protein expressed from yeast, insect cells, bacteria such asE. coli, or other resources by recombinant technology.

Immunization

In some embodiments, human antibodies are produced by immunizing anon-human, transgenic animal comprising within its genome some or all ofhuman immunoglobulin heavy chain and light chain loci with a ALK-1antigen. In a preferred embodiment, the non-human animal is a XENOMOUSE®animal. (Abgenix, Inc., Fremont, Calif.).

XENOMOUSE® mice are engineered mouse strains that comprise largefragments of human immunoglobulin heavy chain and light chain loci andare deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598,5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364,6,162,963 and 6,150,584. See also WO91/10741, WO 94/02602, WO 96/34096,WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO99/53049, WO 00/09560, and WO 00/037504.

In another aspect, the invention provides a method for making anti-ALK-1antibodies from non-human, non-mouse animals by immunizing non-humantransgenic animals that comprise human immunoglobulin loci with an ALK-1antigen. One can produce such animals using the methods described in theabove-cited documents. The methods disclosed in these documents can bemodified as described in U.S. Pat. No. 5,994,619, which is herebyincorporated by reference. U.S. Pat. No. 5,994,619 describes methods forproducing novel cultured inner cell mass (CICM) cells and cell lines,derived from pigs and cows, and transgenic CICM cells into whichheterologous DNA has been inserted. CICM transgenic cells can be used toproduce cloned transgenic embryos, fetuses, and offspring. The '619patent also describes methods of producing transgenic animals that arecapable of transmitting the heterologous DNA to their progeny. Inpreferred embodiments of the current invention, the non-human animalsare mammals, particularly rats, sheep, pigs, goats, cattle, horses orchickens.

XENOMOUSE® mice produce an adult-like human repertoire of fully humanantibodies and generate antigen-specific human antibodies. In someembodiments, the XENOMOUSE® mice contain approximately 80% of the humanantibody V gene repertoire through introduction of megabase sized,germline configuration fragments of the human heavy chain loci and kappalight chain loci in yeast artificial chromosome (YAC). In otherembodiments, XENOMOUSE® mice further contain approximately all of thehuman lambda light chain locus. See Mendez et al., Nature Genetics15:146-156 (1997), Green and Jakobovits, J. Exp. Med. 188:483-495(1998), and WO 98/24893, the disclosures of which are herebyincorporated by reference.

In some embodiments, the non-human animal comprising humanimmunoglobulin genes are animals that have a human immunoglobulin“minilocus”. In the minilocus approach, an exogenous Ig locus ismimicked through the inclusion of individual genes from the Ig locus.Thus, one or more V_(H) genes, one or more D_(H) genes, one or moreJ_(H) genes, a mu constant domain, and a second constant domain(preferably a gamma constant domain) are formed into a construct forinsertion into an animal. This approach is described, inter alia, inU.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425,5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205,5,721,367, 5,789,215, and 5,643,763, hereby incorporated by reference.

In another aspect, the invention provides a method for making humanizedanti-ALK-1 antibodies. In some embodiments, non-human animals areimmunized with a ALK-1 antigen as described below under conditions thatpermit antibody production. Antibody-producing cells are isolated fromthe animals, and nucleic acids encoding the heavy and light chains of ananti-ALK-1 antibody of interest are isolated from the isolatedantibody-producing cells or from an immortalized cell line produced fromsuch cells. These nucleic acids are subsequently engineered usingtechniques known to those of skill in the art and as described furtherbelow to reduce the amount of non-human sequence, i.e., to humanize theantibody to reduce the immune response in humans.

Immunization of animals can be by any method known in the art. See,e.g., , Harlow and Lane Antibodies: A Laboratory Manual, New York: ColdSpring Harbor Press, 1990. Methods for immunizing non-human animals suchas mice, rats, sheep, goats, pigs, cattle and horses are well known inthe art. See, e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619.In a preferred embodiment, the ALK-1 antigen is administered with anadjuvant to stimulate the immune response. Exemplary adjuvants includecomplete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) orISCOM (immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in a local deposit,or they may contain substances that stimulate the host to secretefactors that are chemotactic for macrophages and other components of theimmune system. Preferably, if a polypeptide is being administered, theimmunization schedule will involve two or more administrations of thepolypeptide, spread out over several weeks. Example 2 exemplifies amethod for producing anti-ALK-1 monoclonal antibodies in XENOMOUSE®mice.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with a ALK-1 antigen, antibodies and/orantibody-producing cells can be obtained from the animal. In someembodiments, anti-ALK-1 antibody-containing serum is obtained from theanimal by bleeding or sacrificing the animal. The serum may be used asit is obtained from the animal, an immunoglobulin fraction may beobtained from the serum, or the anti-ALK-1 antibodies may be purifiedfrom the serum.

In some embodiments, antibody-producing cell lines are prepared fromcells isolated from the immunized animal. After immunization, the animalis sacrificed and lymph node and/or splenic B cells are immortalized byany means known in the art. Methods of immortalizing cells include, butare not limited to, transfecting them with oncogenes, infecting themwith an oncogenic virus and cultivating them under conditions thatselect for immortalized cells, subjecting them to carcinogenic ormutating compounds, fusing them with an immortalized cell, e.g., amyeloma cell, and inactivating a tumor suppressor gene. See, e.g.,Harlow and Lane, supra. If fusion with myeloma cells is used, themyeloma cells preferably do not secrete immunoglobulin polypeptides (anon-secretory cell line). Immortalized cells are screened using ALK-1,or a portion thereof. In a preferred embodiment, the initial screeningis performed using an enzyme-linked immunoassay (ELISA) or aradioimmunoassay. An example of ELISA screening is provided in WO00/37504, incorporated herein by reference.

Anti-ALK-1 antibody-producing cells, e.g., hybridomas, are selected,cloned and further screened for desirable characteristics, includingrobust growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas can be expandedin vivo in syngeneic animals, in animals that lack an immune system,e.g., nude mice, or in cell culture in vitro. Methods of selecting,cloning and expanding hybridomas are well known to those of ordinaryskill in the art.

In a preferred embodiment, the immunized animal is a non-human animalthat expresses human immunoglobulin genes and the splenic B cells arefused to a myeloma cell line from the same species as the non-humananimal. In a more preferred embodiment, the immunized animal is aXENOMOUSE® mouse and the myeloma cell line is a non-secretory mousemyeloma. In an even more preferred embodiment, the myeloma cell line isP3-X63-Ag8.653 (American Type Culture Collection). See, e.g., Example 2.

Thus, in one embodiment, the invention provides methods for producing acell line that produces a human monoclonal antibody or a fragmentthereof directed to ALK-1 comprising (a) immunizing a non-humantransgenic animal described herein with ALK-1, a portion of ALK-1 or acell or tissue expressing ALK-1; (b) allowing the transgenic animal tomount an immune response to ALK-1; (c) isolating antibody-producingcells from transgenic animal; (d) immortalizing the antibody-producingcells; (e) creating individual monoclonal populations of theimmortalized antibody-producing cells; and (f) screening theimmortalized antibody-producing cells to identify an antibody directedto ALK-1.

In another aspect, the invention provides a cell line that produces ahuman anti-ALK-1 antibody. In some embodiments the cell line is ahybridoma cell line. In some embodiments, the hybridomas are mousehybridomas, as described above. In other embodiments, the hybridomas areproduced in a non-human, non-mouse species such as rats, sheep, pigs,goats, cattle or horses. In another embodiment, the hybridomas are humanhybridomas.

In another embodiment, a transgenic animal is immunized with an ALK-1antigen, primary cells, e.g., spleen or peripheral blood B cells, areisolated from an immunized transgenic animal and individual cellsproducing antibodies specific for the desired antigen are identified.Polyadenylated mRNA from each individual cell is isolated and reversetranscription polymerase chain reaction (RT-PCR) is performed usingsense primers that anneal to variable domain sequences, e.g., degenerateprimers that recognize most or all of the FR1 regions of human heavy andlight chain variable domain genes and anti-sense primers that anneal toconstant or joining region sequences. cDNAs of the heavy and light chainvariable domains are then cloned and expressed in any suitable hostcell, e.g., a myeloma cell, as chimeric antibodies with respectiveimmunoglobulin constant regions, such as the heavy chain and κ or λconstant domains. See Babcook, J. S. et al., Proc. Natl. Acad. Sci. USA93:7843-48, 1996, incorporated herein by reference. Anti ALK-1antibodies may then be identified and isolated as described herein.

In another embodiment, phage display techniques can be used to providelibraries containing a repertoire of antibodies with varying affinitiesfor ALK-1. For production of such repertoires, it is unnecessary toimmortalize the B cells from the immunized animal. Rather, the primary Bcells can be used directly as a source of DNA. The mixture of cDNAsobtained from B cell, e.g., derived from spleens, is used to prepare anexpression library, for example, a phage display library transfectedinto E. coli. The resulting cells are tested for immunoreactivity toALK-1. Techniques for the identification of high affinity humanantibodies from such libraries are described by Griffiths et al., EMBOJ., 13:3245-3260 (1994); Nissim et al., ibid, pp. 692-698 and byGriffiths et al., ibid, 12:725-734, which are incorporated by reference.Ultimately, clones from the library are identified that produce bindingaffinities of a desired magnitude for the antigen and the DNA encodingthe product responsible for such binding is recovered and manipulatedfor standard recombinant expression. Phage display libraries may also beconstructed using previously manipulated nucleotide sequences andscreened in a similar fashion. In general, the cDNAs encoding heavy andlight chains are independently supplied or linked to form Fv analogs forproduction in the phage library.

The phage library is then screened for the antibodies with the highestaffinities for ALK-1 and the genetic material recovered from theappropriate clone. Further rounds of screening can increase affinity ofthe original antibody isolated.

Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of MakingAntibodies Nucleic Acids

The present invention also encompasses nucleic acid molecules encodinganti-ALK-1 antibodies or an antigen-binding fragments thereof. In someembodiments, different nucleic acid molecules encode a heavy chain and alight chain of an anti-ALK-1 immunoglobulin. In other embodiments, thesame nucleic acid molecule encodes a heavy chain and a light chain of ananti-ALK-1 immunoglobulin.

In some embodiments, the nucleic acid molecule encoding the variabledomain of the light chain (V_(L)) utilizes a human A27, A2, A1, A3, B3,B2, L1 or L2 V_(κ) gene, and a human Jκ5, Jκ1, Jκ3 or Jκ4 gene. In someembodiments the nucleic acid molecule utilizes a human A27 Vκ gene and ahuman Jκ5 gene. In other embodiments, the nucleic acid molecule utilizesa human A2 gene and a human Jκ1 gene. In some embodiments, the nucleicacid molecule encoding the light chain encodes an amino acid sequencecomprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15substitutions from the germline amino acid sequence(s). In someembodiments, the nucleic acid molecule comprises a nucleotide sequencethat encodes a V_(L) amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14 or 15 conservative amino acid substitutionsand/or 1, 2, or 3 non-conservative substitutions compared to germlineV_(κ) and J_(κ) sequences. Substitutions may be in the CDR regions, theframework regions, or in the constant domain.

In some embodiments, the nucleic acid molecule encodes a V_(L) aminoacid sequence comprising one or more mutations compared to the germlinesequence that are identical to the mutations from germline found in theV_(L) of any one of antibodies 1.11.1; 1.12.1; 1.12.1(rWT);1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1;1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1;4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1;5.57.1; or 5.59.1.

In some embodiments, the nucleic acid molecule encodes at least threeamino acid substitutions compared to the germline sequence that areidentical to the mutations from germline found in the V_(L) of any oneof antibodies 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A);1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1;1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1;4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or5.59.1.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence selected from the group consisting of SEQ ID NOs: 7, 11, 15,19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87,91 or 126, which encodes the V_(L) amino acid sequence of monoclonalantibody 1.12.1(M29I/D19A), 1.11.1, 1.13.1; 1.14.1; 1.151.1; 1.162.1;1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1;4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1;5.59.1 or 1.12.1.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of one of SEQ ID NOs: 8;12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72; 76; 80;84; 88; 92 or 127. In some embodiments, the nucleic acid moleculecomprises the nucleotide sequence of SEQ ID NO: 3 or a portion thereof.In some embodiments, the nucleic acid encodes the amino acid sequence ofthe light chain of one, two or all three CDRs of said antibody. In someembodiments, said portion encodes a contiguous region from CDR1-CDR3 ofthe light chain of an anti-ALK-1 antibody.

In some embodiments, the nucleic acid molecule encodes a V_(L) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the V_(L) amino acid sequence of any one of antibodies1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I);1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1;1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1;4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1, or to theamino acid sequence of the V_(L) region of SEQ ID NO: 4. Nucleic acidmolecules of the invention include nucleic acids that hybridize underhighly stringent conditions, such as those described above, or that areat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to anucleic acid encoding the amino acid sequence the V_(L) region of SEQ IDNOs: 8; 12; 16; 20; 24; 28; 32; 36; 40; 44; 48; 52; 56; 60; 64; 68; 72;76; 80; 84; 88; 92 or 126 or to a nucleic acid comprising the V_(L)region nucleotide sequence of SEQ ID NO: 4.

In other preferred embodiments, the nucleic acid molecule encodes thevariable domain of a heavy chain (V_(H)) that utilizes a human V_(H)4-31, V_(H) 3-11, V_(H) 3-15, V_(H) 3-33, V_(H) 4-61 or V_(H) 4-59 genesequence or a sequence derived therefrom. In some embodiments, thenucleic acid molecule utilizes a human V_(H) 4-31 gene, a DH6-19 geneand a human JH4B gene.

In some embodiments, the nucleic acid molecule encodes an amino acidsequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mutationscompared to the germline amino acid sequence of the human V, D or Jgenes. In some embodiments, said mutations are in the V_(H) region. Insome embodiments, said mutations are in the CDR regions.

In some embodiments, the nucleic acid molecule encodes a V_(H) sequencecomprising one or more amino acid mutations compared to the germlineV_(H) sequence that are identical to amino acid mutations found in theV_(H) of any one of monoclonal antibody 1.11.1; 1.12.1; 1.12.1(rWT);1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1;1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1;4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1;5.57.1; or 5.59.1. In some embodiments, the nucleic acid encodes atleast three amino acid mutations compared to the germline sequences thatare identical to at least three amino acid mutations found in one of theabove-listed monoclonal antibodies.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence selected from the group consisting of SEQ ID NOs: 5, 13, 17,21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89,or 103, which encodes the V_(H) amino acid sequence of monoclonalantibody 1.12.1(M29I/D19A), 1.11.1, 1.13.1; 1.14.1; 1.151.1; 1.162.1;1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1;4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1;5.59.1 or 1.12.1.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of one of SEQ ID NOs: SEQID NOs: 2; 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58; 62;66; 70; 74; 78; 82; 86; 90 or 104. In various preferred embodiments, thenucleic acid molecule comprises at least a portion of the nucleotidesequence of SEQ ID NOS: 1 or 95. In some embodiments, said portionencodes the V_(H) region, a CDR3 region, all three CDR regions, or acontiguous region including CDR1-CDR3.

In some embodiments, the nucleic acid molecule encodes a V_(H) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the V_(H) amino acid sequence in any one of SEQ ID NOS:SEQ ID NOs: 2; 6; 10; 14; 18; 22; 26; 30; 34; 38; 42; 46; 50; 54; 58;62; 66; 70; 74; 78; 82; 86; 90 or 104. Nucleic acid molecules of theinvention include nucleic acids that hybridize under highly stringentconditions, such as those described above, or that are at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a nucleic acidencoding the amino acid sequence of SEQ ID NOs: 2; 6; 10; 14; 18; 22;26; 30; 34; 38; 42; 46; 50; 54; 58; 62; 66; 70; 74; 78; 82; 86; 90, 100or 104, or to a V_(H) region thereof, or to a nucleic acid comprisingthe nucleotide sequence of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33,37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 95, 103, 128, or129, or the nucleotide sequence that encodes a V_(H) region thereof.

In another embodiment, the nucleic acid encodes a full-length heavychain of an antibody selected from the group consisting of 1.11.1;1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A);1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1;1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1;5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and 5.59.1, or a heavy chaincomprising the amino acid sequence of SEQ ID NO: 2. Further, the nucleicacid may comprise the nucleotide sequence of SEQ ID NOs: 1 or 95.

A nucleic acid molecule encoding the heavy or light chain of ananti-ALK-1 antibody or portions thereof can be isolated from any sourcethat produces such antibody. In various embodiments, the nucleic acidmolecules are isolated from a B cell that expresses an anti-ALK-1antibody isolated from an animal immunized with ALK-1 or from animmortalized cell derived from such a B cell. Methods of isolatingnucleic acids encoding an antibody are well-known in the art. See, e.g.,Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2000). mRNA may be isolated and used to produce cDNA for use in thepolymerase chain reaction (PCR) or cDNA cloning of antibody genes. In apreferred embodiment, the nucleic acid molecule is isolated from ahybridoma that has as one of its fusion partners a cell from a non-humantransgenic animal, said cell producing a human immunoglobulin. In aneven more preferred embodiment, the cell producing human immunoglobulinis isolated from a XENOMOUSE® animal. In another embodiment, the cellproducing the human immunoglobulin is isolated from a non-human,non-mouse transgenic animal, as described above. In another embodiment,the nucleic acid is isolated from a non-human, non-transgenic animal.The nucleic acid molecules isolated from a non-human, non-transgenicanimal may be used, e.g., for humanized antibodies that comprise one ormore amino acid sequences from a human anti-ALK-1 antibody of thepresent invention.

In some embodiments, a nucleic acid encoding a heavy chain of ananti-ALK-1 antibody of the invention can comprise a nucleotide sequenceencoding a V_(H) domain of the invention joined in-frame to a nucleotidesequence encoding a heavy chain constant domain from any source.Similarly, a nucleic acid molecule encoding a light chain of ananti-ALK-1 antibody of the invention can comprise a nucleotide sequenceencoding a V_(L) domain of the invention joined in-frame to a nucleotidesequence encoding a light chain constant domain from any source.

In a further aspect of the invention, nucleic acid molecules encodingthe variable domain of the heavy (V_(H)) and/or light (V_(L)) chains are“converted” to full-length antibody genes. In one embodiment, nucleicacid molecules encoding the V_(H) or V_(L) domains are converted tofull-length antibody genes by insertion into an expression vectoralready encoding heavy chain constant (C_(H)) or light chain constant(C_(L)) domains, respectively, such that the V_(H) segment isoperatively linked to the C_(H) segment(s) within the vector, and/or theV_(L) segment is operatively linked to the C_(L) segment within thevector. In another embodiment, nucleic acid molecules encoding the V_(H)and/or V_(L) domains are converted into full-length antibody genes bylinking, e.g., ligating, a nucleic acid molecule encoding a V_(H) and/orV_(L) domains to a nucleic acid molecule encoding a C_(H) and/or C_(L)domain using standard molecular biological techniques. Nucleic acidsequences of human heavy and light chain immunoglobulin constant domaingenes are known in the art. See, e.g., Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed., NIH Publ. No. 91-3242,1991. Nucleic acid molecules encoding the full-length heavy and/or lightchains may then be expressed from a cell into which they have beenintroduced and the anti-ALK-1 antibody isolated.

The nucleic acid molecules may be used to recombinantly express largequantities of anti-ALK-1 antibodies. The nucleic acid molecules also maybe used to produce chimeric antibodies, bispecific antibodies, singlechain antibodies, immunoadhesins, diabodies, mutated antibodies andantibody derivatives, as described further below. If the nucleic acidmolecules are derived from a non-human, non-transgenic animal, thenucleic acid molecules may be used for antibody humanization, also asdescribed below.

In another embodiment, a nucleic acid molecule of the invention is usedas a probe or PCR primer for a specific antibody sequence. For instance,the nucleic acid can be used as a probe in diagnostic methods or as aPCR primer to amplify regions of DNA that could be used, inter alia, toisolate additional nucleic acid molecules encoding variable domains ofanti-ALK-1 antibodies. In some embodiments, the nucleic acid moleculesare oligonucleotides. In some embodiments, the oligonucleotides are fromhighly variable domains of the heavy and light chains of the antibody ofinterest. In some embodiments, the oligonucleotides encode all or a partof one or more of the CDRs of antibodies 1.11.1; 1.12.1; 1.12.1(rWT);1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1;1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1;4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1;5.57.1; or 5.59.1 or variants thereof as described herein.

Vectors

The invention provides vectors comprising nucleic acid molecules thatencode the heavy chain of an anti-ALK-1 antibody of the invention or anantigen-binding portion thereof. The invention also provides vectorscomprising nucleic acid molecules that encode the light chain of suchantibodies or antigen-binding portion thereof. The invention furtherprovides vectors comprising nucleic acid molecules encoding fusionproteins, modified antibodies, antibody fragments, and probes thereof.

In some embodiments, the anti-ALK-1 antibodies of the invention orantigen-binding portions are expressed by inserting DNAs encodingpartial or full-length light and heavy chains, obtained as describedabove, into expression vectors such that the genes are operativelylinked to necessary expression control sequences such as transcriptionaland translational control sequences. Expression vectors includeplasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV),plant viruses such as cauliflower mosaic virus, tobacco mosaic virus,cosmids, YACs, EBV derived episomes, and the like. The antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevectors. In a preferred embodiment, both genes are inserted into thesame expression vector. The antibody genes are inserted into theexpression vector by standard methods (e.g., ligation of complementaryrestriction sites on the antibody gene fragment and vector, or blunt endligation if no restriction sites are present).

A convenient vector is one that encodes a functionally complete humanC_(H) or C_(L) immunoglobulin sequence, with appropriate restrictionsites engineered so that any V_(H) or V_(L) sequence can easily beinserted and expressed, as described above. In such vectors, splicingusually occurs between the splice donor site in the inserted J regionand the splice acceptor site preceding the human C domain, and also atthe splice regions that occur within the human C_(H) exons.Polyadenylation and transcription termination occur at nativechromosomal sites downstream of the coding regions. The recombinantexpression vector also can encode a signal peptide that facilitatessecretion of the antibody chain from a host cell. The antibody chaingene may be cloned into the vector such that the signal peptide islinked in-frame to the amino terminus of the immunoglobulin chain. Thesignal peptide can be an immunoglobulin signal peptide or a heterologoussignal peptide (i.e., a signal peptide from a non-immunoglobulinprotein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from retroviral LTRs, cytomegalovirus(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (suchas the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus majorlate promoter (AdMLP)), polyoma and strong mammalian promoters such asnative immunoglobulin and actin promoters. For further description ofviral regulatory elements, and sequences thereof, see e.g., U.S. Pat.No. 5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No. 4,968,615.Methods for expressing antibodies in plants, including a description ofpromoters and vectors, as well as transformation of plants is known inthe art. See, e.g., U.S. Pat. No. 6,517,529, incorporated herein byreference. Methods of expressing polypeptides in bacterial cells orfungal cells, e.g., yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, incorporated herein by reference).For example, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. For example, selectable markergenes include the dihydrofolate reductase (DHFR) gene (for use indhfr-host cells with methotrexate selection/amplification), the neo gene(for G418 selection), and the glutamate synthetase gene.

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molecules encoding anti-ALK-1 antibodies and vectorscomprising these nucleic acid molecules can be used for transfection ofa suitable mammalian, plant, bacterial or yeast host cell.Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.Methods of transforming cells are well known in the art. See, e.g., U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporatedherein by reference). Methods of transforming plant cells are well knownin the art, including, e.g., Agrobacterium-mediated transformation,biolistic transformation, direct injection, electroporation and viraltransformation. Methods of transforming bacterial and yeast cells arealso well known in the art.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, inter alia,Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells,293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, babyhamster kidney (BHK) cells, African green monkey kidney cells (COS),human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and anumber of other cell lines. Cell lines of particular preference areselected through determining which cell lines have high expressionlevels. Other cell lines that may be used are insect cell lines, such asSf9 or Sf21 cells. When recombinant expression vectors encoding antibodygenes are introduced into mammalian host cells, the antibodies areproduced by culturing the host cells for a period of time sufficient toallow for expression of the antibody in the host cells or, morepreferably, secretion of the antibody into the culture medium in whichthe host cells are grown. Antibodies can be recovered from the culturemedium using standard protein purification methods. Plant host cellsinclude, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato,etc. Bacterial host cells include E. coli and Streptomyces species.Yeast host cells include Schizosaccharomyces pombe, Saccharomycescerevisiae and Pichia pastoris.

Further, expression of antibodies of the invention from production celllines can be enhanced using a number of known techniques. For example,the glutamine synthetase gene expression system (the GS system) is acommon approach for enhancing expression under certain conditions. TheGS system is discussed in whole or part in connection with EuropeanPatent Nos. 0 216 846, 0 256 055, 0 323 997 and 0 338 841.

It is likely that antibodies expressed by different cell lines or intransgenic animals will have different glycosylation from each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the instant invention, regardless of the glycosylation of theantibodies.

Transgenic Animals and Plants

Anti-ALK-1 antibodies of the invention also can be producedtransgenically through the generation of a mammal or plant that istransgenic for the immunoglobulin heavy and light chain sequences ofinterest and production of the antibody in a recoverable form therefrom.In connection with the transgenic production in mammals, anti-ALK-1antibodies can be produced in, and recovered from, the milk of goats,cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687,5,750,172, and 5,741,957, incorporated herein by reference. In someembodiments, non-human transgenic animals that comprise humanimmunoglobulin loci are immunized with ALK-1 or an immunogenic portionthereof, as described above. Methods for making antibodies in plants aredescribed, e.g., in U.S. Pat. Nos. 6,046,037 and 5,959,177, incorporatedherein by reference.

In some embodiments, non-human transgenic animals or plants are producedby introducing one or more nucleic acid molecules encoding an anti-ALK-1antibody of the invention into the animal or plant by standardtransgenic techniques. See Hogan and U.S. Pat. No. 6,417,429, supra. Thetransgenic cells used for making the transgenic animal can be embryonicstem cells or somatic cells or a fertilized egg. The transgenicnon-human organisms can be chimeric, nonchimeric heterozygotes, andnonchimeric homozygotes. See, e.g., Hogan et al., Manipulating the MouseEmbryo: A Laboratory Manual 2^(nd) ed., Cold Spring Harbor Press (1999);Jackson et al., Mouse Genetics and Transgenics: A Practical Approach,Oxford University Press (2000); and Pinkert, Transgenic AnimalTechnology: A Laboratory Handbook, Academic Press (1999), allincorporated herein by reference. In some embodiments, the transgenicnon-human animals have a targeted disruption and replacement by atargeting construct that encodes a heavy chain and/or a light chain ofinterest. In a preferred embodiment, the transgenic animals comprise andexpress nucleic acid molecules encoding heavy and light chains thatspecifically bind to ALK-1, preferably human ALK-1. In some embodiments,the transgenic animals comprise nucleic acid molecules encoding amodified antibody such as a single-chain antibody, a chimeric antibodyor a humanized antibody. The anti-ALK-1 antibodies may be made in anytransgenic animal. In a preferred embodiment, the non-human animals aremice, rats, sheep, pigs, goats, cattle or horses. The non-humantransgenic animal expresses said encoded polypeptides in blood, milk,urine, saliva, tears, mucus and other bodily fluids.

Phage Display Libraries

The invention provides a method for producing an anti-ALK-1 antibody orantigen-binding portion thereof comprising the steps of synthesizing alibrary of human antibodies on phage, screening the library with ALK-1or an antibody-binding portion thereof, isolating phage that bind ALK-1,and obtaining the antibody from the phage. By way of example, one methodfor preparing the library of antibodies for use in phage displaytechniques comprises the steps of immunizing a non-human animalcomprising human immunoglobulin loci with ALK-1 or an antigenic portionthereof to create an immune response, extracting antibody-producingcells from the immunized animal; isolating RNA encoding heavy and lightchains of antibodies of the invention from the extracted cells, reversetranscribing the RNA to produce cDNA, amplifying the cDNA using primers,and inserting the cDNA into a phage display vector such that antibodiesare expressed on the phage. Recombinant anti-ALK-1 antibodies of theinvention may be obtained in this way.

Recombinant human anti-ALK-1 antibodies of the invention can be isolatedby screening a recombinant combinatorial antibody library. Preferablythe library is a scFv phage display library, generated using human V_(L)and V_(H) cDNAs prepared from mRNA isolated from B cells. Methods forpreparing and screening such libraries are known in the art. Kits forgenerating phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; andthe Stratagene SurfZAP™ phage display kit, catalog no. 240612). Therealso are other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., U.S. Pat. No.5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791,WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al.,Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); McCafferty etal., Nature 348:552-554 (1990); Griffiths et al., EMBO J. 12:725-734(1993); Hawkins et al., J. Mol. Biol. 226:889-896 (1992); Clackson etal., Nature 352:624-628 (1991); Gram et al., Proc. Natl.Acad. Sci. USA89:3576-3580 (1992); Garrad et al., Bio/Technology 9:1373-1377 (1991);Hoogenboom et al., Nuc. Acid Res. 19:4133-4137 (1991); and Barbas etal., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991), all incorporatedherein by reference.

In one embodiment, to isolate and produce human anti-ALK-1 antibodieswith the desired characteristics, a human anti-ALK-1 antibody asdescribed herein is first used to select human heavy and light chainsequences having similar binding activity toward ALK-1, using theepitope imprinting methods described in PCT Publication No. WO 93/06213,incorporated herein by reference. The antibody libraries used in thismethod are preferably scFv libraries prepared and screened as describedin PCT Publication No. WO 92/01047, McCafferty et al., Nature348:552-554 (1990); and Griffiths et al., EMBO J. 12:725-734 (1993), allincorporated herein by reference. The scFv antibody libraries preferablyare screened using human ALK-1 as the antigen.

Once initial human V_(L) and V_(H) domains are selected, “mix and match”experiments are performed, in which different pairs of the initiallyselected V_(L) and V_(H) segments are screened for ALK-1 binding toselect preferred V_(L)/V_(H) pair combinations. Additionally, to furtherimprove the quality of the antibody, the V_(L) and V_(H) segments of thepreferred V_(L)/V_(H) pair(s) can be randomly mutated, preferably withinthe CDR3 region of V_(H) and/or V_(L), in a process analogous to the invivo somatic mutation process responsible for affinity maturation ofantibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying V_(H) and V_(L) domainsusing PCR primers complimentary to the V_(H) CDR3 or V_(L) CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be re-screened for bindingto ALK-1.

Following screening and isolation of an anti-ALK-1 antibody of theinvention from a recombinant immunoglobulin display library, nucleicacids encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can further be manipulated to create other antibodyforms of the invention, as described below. To express a recombinanthuman antibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described above.

Deimmunized Antibodies

In another aspect of the invention, the antibody may be deimmunized toreduce its immunogenicity using the techniques described in, e.g., PCTPublication Nos. WO98/52976 and WO00/34317 (incorporated herein byreference).

Mutated Antibodies

In another embodiment, the nucleic acid molecules, vectors and hostcells may be used to make mutated anti-ALK-1 antibodies. The antibodiesmay be mutated in the variable domains of the heavy and/or light chains,e.g., to alter a binding property of the antibody. For example, amutation may be made in one or more of the CDR regions to increase ordecrease the K_(D) of the antibody for ALK-1, to increase or decreasek_(off), or to alter the binding specificity of the antibody. Techniquesin site-directed mutagenesis are well-known in the art. See, e.g.,Sambrook et al. and Ausubel et al., supra. In another embodiment, one ormore mutations are made at an amino acid residue that is known to bechanged compared to the germline in monoclonal antibody 1.11.1; 1.12.1;1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A); 1.13.1;1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1; 1.8.1; 1.9.1;4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1; 5.13.1; 5.34.1;5.53.1; 5.56.1; 5.57.1; or 5.59.1. The mutations may be made in a CDRregion or framework region of a variable domain, or in a constantdomain. In a preferred embodiment, the mutations are made in a variabledomain. In some embodiments, one or more mutations are made at an aminoacid residue that is known to be changed compared to the germline in aCDR region or framework region of a variable domain of an amino acidsequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90 92 or 127, or whosenucleic acid sequence is presented in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 95, 102 or 126.

In another embodiment, the framework region is mutated so that theresulting framework region(s) have the amino acid sequence of thecorresponding germline gene. A mutation may be made in a frameworkregion or constant domain to increase the half-life of the anti-ALK-1antibody. See, e.g., PCT Publication No. WO 00/09560, incorporatedherein by reference. A mutation in a framework region or constant domainalso can be made to alter the immunogenicity of the antibody, to providea site for covalent or non-covalent binding to another molecule, or toalter such properties as complement fixation, FcR binding andantibody-dependent cell-mediated cytotoxicity (ADCC). According to theinvention, a single antibody may have mutations in any one or more ofthe CDRs or framework regions of the variable domain or in the constantdomain.

In some embodiments, there are from 1 to 13, including any number inbetween, amino acid mutations in either the V_(H) or V_(L) domains ofthe mutated anti-ALK-1 antibody compared to the anti-ALK-1 antibodyprior to mutation. In any of the above, the mutations may occur in oneor more CDR regions. Further, any of the mutations can be conservativeamino acid substitutions. In some embodiments, there are no more than 5,4, 3, 2, or 1 amino acid changes in the constant domains.

Modified Antibodies

In another embodiment, a fusion antibody or immunoadhesin may be madethat comprises all or a portion of an anti-ALK-1 antibody of theinvention linked to another polypeptide. In a preferred embodiment, onlythe variable domains of the anti-ALK-1 antibody are linked to thepolypeptide. In another preferred embodiment, the V_(H) domain of ananti-ALK-1 antibody is linked to a first polypeptide, while the V_(L)domain of an anti-ALK-1 antibody is linked to a second polypeptide thatassociates with the first polypeptide in a manner such that the V_(H)and V_(L) domains can interact with one another to form an antigenbinding site. In another preferred embodiment, the V_(H) domain isseparated from the V_(L) domain by a linker such that the V_(H) andV_(L) domains can interact with one another (see below under SingleChain Antibodies). The V_(H)-linker-V_(L) antibody is then linked to thepolypeptide of interest. In addition, fusion antibodies can be createdin which two (or more) single-chain antibodies are linked to oneanother. This is useful if one wants to create a divalent or polyvalentantibody on a single polypeptide chain, or if one wants to create abispecific antibody.

To create a single chain antibody, (scFv) the V_(H)- and V_(L)-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃,such that the V_(H) and V_(L) sequences can be expressed as a contiguoussingle-chain protein, with the V_(L) and V_(H) domains joined by theflexible linker. See, e.g., Bird et al., Science 242:423-426 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);McCafferty et al., Nature 348:552-554 (1990). The single chain antibodymay be monovalent, if only a single V_(H) and V_(L) are used, bivalent,if two V_(H) and V_(L) are used, or polyvalent, if more than two V_(H)and V_(L) are used. Bispecific or polyvalent antibodies may be generatedthat bind specifically to ALK-1 and to another molecule.

In other embodiments, other modified antibodies may be prepared usinganti-ALK-1 antibody encoding nucleic acid molecules. For instance,“Kappa bodies” (Ill et al., Protein Eng. 10: 949-57 (1997)),“Minibodies” (Martin et al., EMBO J. 13: 5303-9 (1994)), “Diabodies”(Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or“Janusins” (Traunecker et al., EMBO J. 10:3655-3659 (1991) andTraunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) may beprepared using standard molecular biological techniques following theteachings of the specification.

Bispecific antibodies or antigen-binding fragments can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992). Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some embodiments, the bispecific antibody binds to twodifferent epitopes of ALK-1. In some embodiments, the bispecificantibody has a first heavy chain and a first light chain from monoclonalantibody 1.11.1; 1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I);1.12.1(D19A); 1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1;1.31.1; 1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1;4.72.1; 5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; or 5.59.1 and anadditional antibody heavy chain and light chain. In some embodiments,the additional light chain and heavy chain also are from one of theabove-identified monoclonal antibodies, but are different from the firstheavy and light chains.

In some embodiments, the modified antibodies described above areprepared using one or more of the variable domains or CDR regions from ahuman anti-ALK-1 monoclonal antibody provided herein.

Derivatized and Labeled Antibodies

An anti-ALK-1 antibody or antigen-binding portion of the invention canbe derivatized or linked to another molecule (e.g., another peptide orprotein). In general, the antibodies or portion thereof are derivatizedsuch that the ALK-1 binding is not affected adversely by thederivatization or labeling. Accordingly, the antibodies and antibodyportions of the invention are intended to include both intact andmodified forms of the human anti-ALK-1 antibodies described herein. Forexample, an antibody or antibody portion of the invention can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (e.g., a bispecific antibody or a diabody), adetection agent, a pharmaceutical agent, and/or a protein or peptidethat can mediate association of the antibody or antibody portion withanother molecule (such as a streptavidin core region or a polyhistidinetag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Another type of derivatized antibody is a labeled antibody. Usefuldetection agents with which an antibody or antigen-binding portion ofthe invention may be derivatized include fluorescent compounds,including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. An antibody can also be labeled with enzymesthat are useful for detection, such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase andthe like. When an antibody is labeled with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce areaction product that can be discerned. For example, when the agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody can also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Anantibody can also be labeled with a predetermined polypeptide epitoperecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

An anti-ALK-1 antibody can also be derivatized with a chemical groupsuch as polyethylene glycol (PEG), a methyl or ethyl group, or acarbohydrate group. These groups are useful to improve the biologicalcharacteristics of the antibody, e.g., to increase serum half-life.

Pharmaceutical Compositions and Administration

This invention also relates to a pharmaceutical composition for thetreatment of conditions associated with undesirable increasedangiogenesis in a mammal, including a human, comprising an amount of ananti-ALK-1 antibody or antigen binding portion thereof, as describedherein, that is effective in treating such conditions, and apharmaceutically acceptable carrier.

The antibodies and antigen-binding portions of the present invention canbe incorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antigen-binding portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” means any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Someexamples of pharmaceutically acceptable carriers are water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Additionalexamples of pharmaceutically acceptable substances are wetting agents orminor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the antibody.

The compositions of this invention may be in a variety of forms, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Thepreferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans. The preferred mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In a preferred embodiment, the antibodyis administered by intravenous infusion or injection. In anotherpreferred embodiment, the antibody is administered by intramuscular orsubcutaneous injection. Formulations for injection may be presented inunit dosage form, e.g., in ampoules or in multi-dose containers, with orwithout an added preservative. The compositions may take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Alternatively, the active ingredient may be in powderform for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the anti-ALK-1 antibody inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The antibodies or antibody portions of the present invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration issubcutaneous, intramuscular, or intravenous infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results.

In certain embodiments, the antibody compositions of the presentinvention may be prepared with a carrier that will protect the antibodyagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are generally known to those skilled in the art.See, e.g., Sustained and Controlled Release Drug Delivery Systems J. R.Robinson, ed., Marcel Dekker, Inc., New York, 1978, which isincorporated herein by reference.

Additional active compounds also can be incorporated into thecompositions. In certain embodiments, an inhibitory anti-ALK-1 antibodyof the invention is co-formulated with and/or co-administered with oneor more additional therapeutic agents. These agents include, withoutlimitation, antibodies that bind other targets, anti-tumor agents,anti-angiogenesis agents, signal transduction inhibitors,anti-proliferative agents, chemotherapeutic agents, or peptide analoguesthat inhibit anti-ALK-1. Such combination therapies may require lowerdosages of the inhibitory anti-ALK-1 antibody as well as theco-administered agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies.

As noted above, the compositions of the present invention optionally mayfurther comprise a pharmaceutically acceptable antioxidant in additionto a chelating agent. Suitable antioxidants include, but are not limitedto, methionine, sodium thiosulfate, catalase, and platinum. For example,the composition may contain methionine in a concentration that rangesfrom 1 mM to about 100 mM, and in particular, is about 27 mM. Forexample, an aqueous formulation may be: 10 mg/mL anti-ALK-1 antibody, 20mM Histidine, pH 5.5, 84 mg/mL Trehalose dihydrate, 0.2 mg/mLPolysorbate 80, 0.05 mg/mL disodium EDTA, 0.1 mg/mL L-Methionine.

The compositions of the invention may include a “therapeuticallyeffective amount” or a “prophylactically effective amount” of anantibody or antigen-binding portion of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the antibody or antigen-bindingportion may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the antibody orantibody portion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antigen-binding portion areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount may be less than the therapeuticallyeffective amount.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses can be administeredover time or the dose can be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the anti-ALK-1 antibody or portion thereof and theparticular therapeutic or prophylactic effect to be achieved, and (b)the limitations inherent in the art of compounding such an antibody forthe treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg,more preferably 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg. In someembodiments, a formulation contains 5 mg/mL of antibody in a buffer of20 mM sodium citrate, pH 5.5, 140 mM NaCl, and 0.2 mg/mL polysorbate 80.It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

Another aspect of the present invention provides kits comprising ananti-ALK-1 antibody or antigen-binding portion of the invention or acomposition comprising such an antibody or portion. A kit may include,in addition to the antibody or composition, diagnostic or therapeuticagents. A kit can also include instructions for use in a diagnostic ortherapeutic method. In a preferred embodiment, the kit includes theantibody or a composition comprising it and a diagnostic agent that canbe used in a method described below. In another preferred embodiment,the kit includes the antibody or a composition comprising it and one ormore therapeutic agents that can be used in a method described below.

Diagnostic Methods of Use

The anti-ALK-1 antibodies or antigen-binding portions thereof can beused in diagnostic methods to detect ALK-1 in a biological sample invitro or in vivo. For example, the anti-ALK-1 antibodies can be used ina conventional immunoassay, including, without limitation, an ELISA, anRIA, flow cytometry, tissue immunohistochemistry, Western blot orimmunoprecipitation. The anti-ALK-1 antibodies of the invention can beused to detect ALK-1 from humans. The anti-ALK-1 antibodies can also beused to detect ALK-1 from other primates, e.g. cynomolgus monkeys.

The invention provides a method for detecting ALK-1 in a biologicalsample comprising contacting the biological sample with an anti-ALK-1antibody of the invention and detecting the bound antibody. In oneembodiment, the anti-ALK-1 antibody is directly labeled with adetectable label. In another embodiment, the anti-ALK-1 antibody (thefirst antibody) is unlabeled and a second antibody or other moleculethat can bind the anti-ALK-1 antibody is labeled. As is well known toone of skill in the art, a second antibody is chosen that is able tospecifically bind the particular species and class of the firstantibody. For example, if the anti-ALK-1 antibody is a human IgG, thenthe secondary antibody could be an anti-human-IgG. Other molecules thatcan bind to antibodies include, without limitation, Protein A andProtein G, both of which are available commercially, e.g., from PierceChemical Co.

Suitable labels for the antibody or secondary antibody have beendiscussed previously, and include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In other embodiments, ALK-1 can be assayed in a biological sample by acompetition immunoassay utilizing ALK-1 standards labeled with adetectable substance and an unlabeled anti-ALK-1 antibody. In thisassay, the biological sample, the labeled ALK-1 standards and theanti-ALK-1 antibody are combined and the amount of labeled ALK-1standard bound to the unlabeled antibody is determined. The amount ofALK-1 in the biological sample is inversely proportional to the amountof labeled ALK-1 standard bound to the anti-ALK-1 antibody.

One can use the immunoassays disclosed above for a number of purposes.For example, the anti-ALK-1 antibodies can be used to detect ALK-1 incultured cells. In a preferred embodiment, the anti-ALK-1 antibodies areused to determine the amount of ALK-1 produced by cells that have beentreated with various compounds. This method can be used to identifycompounds that modulate ALK-1 protein levels. According to this method,one sample of cells is treated with a test compound for a period of timewhile another sample is left untreated. If the total level of ALK-1 isto be measured, the cells are lysed and the total ALK-1 level ismeasured using one of the immunoassays described above. The total levelof ALK-1 in the treated versus the untreated cells is compared todetermine the effect of the test compound.

A preferred immunoassay for measuring total ALK-1 levels is flowcytometry or immunohistochemistry. Methods such as ELISA, RIA, flowcytometry, Western blot, immunohistochemistry, cell surface labeling ofintegral membrane proteins and immunoprecipitation are well known in theart. See, e.g., Harlow and Lane, supra. In addition, the immunoassayscan be scaled up for high throughput screening in order to test a largenumber of compounds for either activation or inhibition of ALK-1expression.

The anti-ALK-1 antibodies of the invention also can be used to determinethe levels of ALK-1 in a tissue or in cells derived from the tissue. Insome embodiments, the tissue is a diseased tissue. In some embodimentsof the method, a tissue or a biopsy thereof is excised from a patient.The tissue or biopsy is then used in an immunoassay to determine, e.g.,total ALK-1 levels or localization of ALK-1 by the methods discussedabove.

The antibodies of the present invention also can be used in vivo toidentify tissues and organs that express ALK-1. One advantage of usingthe human anti-ALK-1 antibodies of the present invention is that theymay safely be used in vivo without eliciting a substantial immuneresponse to the antibody upon administration, unlike antibodies ofnon-human origin or with humanized or chimeric antibodies.

The method comprises the steps of administering a detectably labeledanti-ALK-1 antibody or a composition comprising them to a patient inneed of such a diagnostic test and subjecting the patient to imaginganalysis to determine the location of the ALK-1-expressing tissues.Imaging analysis is well known in the medical art, and includes, withoutlimitation, x-ray analysis, magnetic resonance imaging (MRI) or computedtomography (CT). The antibody can be labeled with any agent suitable forin vivo imaging, for example a contrast agent, such as barium, which canbe used for x-ray analysis, or a magnetic contrast agent, such as agadolinium chelate, which can be used for MRI or CT. Other labelingagents include, without limitation, radioisotopes, such as ⁹⁹Tc. Inanother embodiment, the anti-ALK-1 antibody will be unlabeled and willbe imaged by administering a second antibody or other molecule that isdetectable and that can bind the anti-ALK-1 antibody. In one embodiment,a biopsy is obtained from the patient to determine whether the tissue ofinterest expresses ALK-1.

Therapeutic Methods of Use

In another embodiment, the invention provides a method for inhibitingALK-1 activity by administering an anti-ALK-1 antibody to a patient inneed thereof. Any of the antibodies or antigen-binding portions thereofdescribed herein may be used therapeutically. In a preferred embodiment,the anti-ALK-1 antibody is a human, chimeric or humanized antibody. Inanother preferred embodiment, the anti-ALK-1 antibody is human antibody,and the patient is a human patient. Alternatively, the patient may be amammal that expresses ALK-1 that the anti-ALK-1 antibody cross-reactswith. The antibody may be administered to a non-human mammal expressingALK-1 with which the antibody cross-reacts (e.g. a cynomolgus monkey)for veterinary purposes or as an animal model of human disease. Suchanimal models may be useful for evaluating the therapeutic efficacy ofantibodies of this invention.

In another embodiment, an anti-ALK-1 antibody or antibody portionthereof may be administered to a patient who expresses inappropriatelyhigh levels of ALK-1. The antibody may be administered once, but morepreferably is administered multiple times. The antibody may beadministered from three times daily to once every six months or longer.The administering may be on a schedule such as three times daily, twicedaily, once daily, once every two days, once every three days, onceweekly, once every two weeks, once every month, once every two months,once every three months and once every six months. The antibody may alsobe administered continuously via a minipump. The antibody may beadministered via a mucosal, buccal, intranasal, inhalable, intravenous,subcutaneous, intramuscular, parenteral, or intratumor route. Theantibody may be administered once, at least twice or for at least theperiod of time until the condition is treated, palliated or cured. Theantibody generally will be administered for as long as the condition ispresent. The antibody will generally be administered as part of apharmaceutical composition as described supra. The dosage of antibodywill generally be in the range of 0.1 to 100 mg/kg, more preferably 0.5to 50 mg/kg, more preferably 1 to 20 mg/kg, and even more preferably 1to 10 mg/kg. The serum concentration of the antibody may be measured byany method known in the art.

In one embodiment, the antibody is administered in a formulation as asterile aqueous solution having a pH that ranges from about 5.0 to about6.5 and comprising from about 1 mg/ml to about 200 mg/ml of antibody,from about 1 millimolar to about 100 millimolar of histidine buffer,from about 0.01 mg/ml to about 10 mg/ml of polysorbate 80, from about100 millimolar to about 400 millimolar of trehalose, and from about 0.01millimolar to about 1.0 millimolar of disodium EDTA dihydrate.

It is further contemplated by the present invention that any of thecompositions herein may be administered to a patient susceptible to orsuffering from a condition associated with increased angiogenesis (“anangiogenic condition”).

Examples of angiogenic conditions that may be treated/prevented by thecompositions/methods of the present invention include, but are notlimited to, cancer (both solid and hematologic), age-related maculardegeneration (AMD), developmental abnormalities (organogenesis),diabetic blindness, endometriosis, ocular neovascularization, psoriasis,rheumatoid arthritis (RA), and skin disclolorations (e.g., hemangioma,nevus flammeus, or nevus simplex).

For example, the present invention relates to methods for treating orpreventing conditions associated with ocular neovascularization usingany of the compositions/methods herein. Conditions associated withocular neovascularization include, but are not limited to, diabeticretinopathy, age related macular degeneration (“ARMD”), rubeoticglaucoma, interstitial keratitis, retinopathy of prematurity, ischemicretinopathy (e.g., sickle cell), pathological myopic, ocularhistoplasmosis, pterygia, punitiate inner choroidopathy, and the like.

Treatment of Abnormal Cell Growth

This invention also relates to a method for the treatment of abnormalcell growth in a mammal, including a human, comprising administering tosaid mammal a therapeutically effective amount of an anti-ALK-1 antibodyor antigen binding portion thereof, as described herein, that iseffective in treating abnormal cell growth.

In one embodiment of this method, the abnormal cell growth is cancer,including, but not limited to, mesothelioma, hepatobilliary (hepatic andbilliary duct), a primary or secondary CNS tumor, a primary or secondarybrain tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, ovarian cancer, colon cancer, rectal cancer,cancer of the anal region, stomach cancer, gastrointestinal (gastric,colorectal, and duodenal), breast cancer, uterine cancer, carcinoma ofthe fallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin'sDisease, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, prostate cancer, testicularcancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocyticlymphomas, cancer of the bladder, cancer of the kidney or ureter, renalcell carcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), primary CNS lymphoma, non hodgkins's lymphoma,spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocorticalcancer, gall bladder cancer, multiple myeloma, cholangiocarcinoma,fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one ormore of the foregoing cancers.

In a preferred embodiment of the present invention the cancer isselected from lung cancer (NSCLC and SCLC), cancer of the head or neck,ovarian cancer, colon cancer, rectal cancer, cancer of the anal region,stomach cancer, breast cancer, cancer of the kidney or ureter, renalcell carcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), primary CNS lymphoma, non hodgkins's lymphoma,spinal axis tumors, or a combination of one or more of the foregoingcancers.

In another preferred embodiment of the present invention the cancer isselected from lung cancer (NSCLC and SCLC), ovarian cancer, coloncancer, rectal cancer, cancer of the anal region, or a combination ofone or more of the foregoing cancers.

In another embodiment of said method, said abnormal cell growth is abenign proliferative disease, including, but not limited to, psoriasis,benign prostatic hypertrophy or restinosis.

This invention also relates to a method for the treatment of abnormalcell growth in a mammal which comprises administering to said mammal anamount of an anti-ALK-1 antibody or antigen binding portion thereof, asdescribed herein, that is effective in treating abnormal cell growth incombination with an anti-tumor agent selected from the group consistingof mitotic inhibitors, alkylating agents, anti -metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, biological responsemodifiers, antibodies, cytotoxics, anti-hormones, and anti-androgens.

The invention also relates to a pharmaceutical composition for thetreatment of abnormal cell growth in a mammal, including a human, whichcomprises an amount of an anti-ALK-1 antibody or antigen binding portionthereof, as described herein, that is effective in treating abnormalcell growth in combination with a pharmaceutically acceptable carrierand an anti-tumor agent selected from the group consisting of mitoticinhibitors, alkylating agents, anti-metabolites, intercalatingantibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes,topoisomerase inhibitors, biological response modifiers, anti-hormones,and anti-androgens.

The invention also relates to a method for the treatment of ahyperproliferative disorder in a mammal which comprises administering tosaid mammal a therapeutically effective amount of an anti-ALK-1 antibodyor antigen binding portion thereof, as described herein, in combinationwith an anti-tumor agent selected from the group consistingantiproliferative agents, kinase inhibitors, angiogenesis inhibitors,growth factor inhibitors, cox-I inhibitors, cox-II inhibitors, mitoticinhibitors, alkylating agents, anti -metabolites, intercalatingantibiotics, growth factor inhibitors, radiation, cell cycle inhibitors,enzymes, topoisomerase inhibitors, biological response modifiers,antibodies, cytotoxics, anti-hormones, statins, and anti-androgens.

In one embodiment of the present invention the anti-tumor agent used inconjunction with an anti-ALK-1 antibody or antigen binding portionthereof, and pharmaceutical compositions described herein, is ananti-angiogenesis agent, kinase inhibitor, pan kinase inhibitor orgrowth factor inhibitor. Preferred pan kinase inhibitors include Sutent(Pfizer Inc., SU-11248), described in U.S. Pat. No. 6,573,293 (Pfizer,Inc, NY, USA).

Anti-angiogenesis agents, include but are not limited to the followingagents, such as EGF inhibitor, EGFR inhibitors, VEGF inhibitors, VEGFRinhibitors, TIE2 inhibitors, IGF1R inhibitors, COX-II (cyclooxygenaseII) inhibitors, MMP-2 (matrix-metalloprotienase 2) inhibitors, and MMP-9(matrix-metalloprotienase 9) inhibitors. Preferred VEGF inhibitors,include for example, Avastin (bevacizumab), an anti-VEGF monoclonalantibody of Genentech, Inc. of South San Francisco, Calif.

Additional VEGF inhibitors include CP-547,632 (Pfizer Inc., NY, USA),Axitinib (Pfizer Inc.; AG-013736), ZD-6474 (AstraZeneca), AEE788(Novartis), AZD-2171), VEGF Trap (Regeneron/Aventis), Vatalanib (alsoknown as PTK-787, ZK-222584: Novartis & Schering AG), MACUGEN®(pegaptanib sodium, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862(Cytran Inc. of Kirkland, Wash.,USA); and angiozyme, a syntheticribozyme from Ribozyme (Boulder, Colo.) and Chiron (Emeryville, Calif.)and combinations thereof. VEGF inhibitors useful in the practice of thepresent invention are disclosed in U.S. Pat. Nos. 6,534,524 and6,235,764, both of which are incorporated in their entirety for allpurposed. Particularly preferred VEGF inhibitors include CP-547,632,AG13736, Vatalanib, MACUGEN® and (pegaptanib sodium) combinationsthereof.

Additional VEGF inhibitors are described in, for example in WO 99/24440(published May 20, 1999), PCT International Application PCT/IB99/00797(filed May 3, 1999), in WO 95/21613 (published Aug. 17, 1995), WO99/61422 (published Dec. 2, 1999), U.S. Pat. No. 6,534,524 (disclosesAG13736), U.S. Pat. No. 5,834,504 (issued Nov. 10, 1998), WO 98/50356(published Nov. 12, 1998), U.S. Pat. No. 5,883,113 (issued Mar. 16,1999), U.S. Pat. No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat. No.5,792,783 (issued Aug. 11, 1998), U.S. Pat. No. 6,653,308 (issued Nov.25, 2003), WO 99/10349 (published Mar. 4, 1999), WO 97/32856 (publishedSep. 12, 1997), WO 97/22596 (published Jun. 26, 1997), WO 98/54093(published Dec. 3, 1998), WO 98/02438 (published Jan. 22, 1998), WO99/16755 (published Apr. 8, 1999), and WO 98/02437 (published Jan. 22,1998), all of which are herein incorporated by reference in theirentirety.

Other antiproliferative agents that may be used with the antibodies, orantigen-binding portions thereof, of the present invention includeinhibitors of the enzyme farnesyl protein transferase and inhibitors ofthe receptor tyrosine kinase PDGFr, including the compounds disclosedand claimed in the following United States patent applications: Ser. No.09/221,946 (filed Dec. 28, 1998); Ser. No. 09/454,058 (filed Dec. 2,1999); Ser. No. 09/501,163 (filed Feb. 9, 2000); Ser. No. 09/539,930(filed Mar. 31, 2000); Ser. No. 09/202,796 (filed May 22, 1997); Ser.No. 09/384,339 (filed Aug. 26, 1999); and Ser. No. 09/383,755 (filedAug. 26, 1999); and the compounds disclosed and claimed in the followingUnited States provisional patent applications: 60/168,207 (filed Nov.30, 1999); 60/170,119 (filed Dec. 10, 1999); 60/177,718 (filed Jan. 21,2000); 60/168,217 (filed Nov. 30, 1999), and 60/200,834 (filed May 1,2000). Each of the foregoing patent applications and provisional patentapplications is herein incorporated by reference in their entirety.

For additional PDGRr inhibitors, see WO01/40217, published Jul. 7, 2001and WO2004/020431, published Mar. 11, 2004, the contents of which areincorporated in their entirety for all purposes. Preferred PDGFrinhibitors include Pfizer's CP-868,596 and its pharmaceuticallyacceptable salts.

Preferred GARF inhibitors include Pfizer's AG-2037 (pelitrexol and itspharmaceutically acceptable salts). GARF inhibitors useful in thepractice of the present invention are disclosed in U.S. Pat. No.5,608,082 which is incorporated in its entirety for all purposes.

Examples of useful COX-II inhibitors which can be used in conjunctionwith a Anti-ALK-1 antibody or antigen binding portion thereof, asdescribed herein, and pharmaceutical compositions described hereininclude CELEBREX® (celecoxib), parecoxib, deracoxib, ABT-963, MK-663(etoricoxib), COX-189 (Lumiracoxib), BMS 347070, RS 57067, NS-398,BEXTRA® (valdecoxib), paracoxib, Vioxx (rofecoxib), SD-8381,4-Methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoyl-phenyl)-1H-pyrrole,2-(4-Ethoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl) -1H-pyrrole, T-614,JTE-522, S-2474, SVT-2016, CT-3, SC-58125 and ARCOXIA® (etoricoxib). Foradditonal COX-II inhibitors, see U.S. patent application Ser. Nos.10/801,446 and 10/801,429, the contents of which are incorporated intheir entirety for all purposes.

In one preferred embodiment the anti-tumor agent is celecoxib, see U.S.Pat. No. 5,466,823, the contents of which are incorporated by referencein its entirety for all purposes. The structure for Celecoxib is shownbelow:

In one preferred embodiment the anti-tumor agent is valecoxib, see U.S.Pat. No. 5,633,272, the contents of which are incorporated by referencein its entirety for all purposes. The structure for valdecoxib is shownbelow:

In one preferred embodiment the anti-tumor agent is parecoxib, see U.S.Pat. No. 5,932,598, the contents of which are incorporated by referencein its entirety for all purposes. The structure for paracoxib is shownbelow:

In one preferred embodiment the anti-tumor agent is deracoxib, see U.S.Pat. No. 5,521,207, the contents of which are incorporated by referencein its entirety for all purposes. The structure for deracoxib is shownbelow:

In one preferred embodiment the anti-tumor agent is SD-8381, see U.S.Pat. No. 6,034,256, the contents of which are incorporated by referencein its entirety for all purposes. The structure for SD-8381 is shownbelow:

In one preferred embodiment the anti-tumor agent is ABT-963, seeInternational Publication Number WO 2002/24719, the contents of whichare incorporated by reference in its entirety for all purposes. Thestructure for ABT-963 is shown below:

In one preferred embodiment the anti-tumor agent is rofecoxib as shownbelow:

In one preferred embodiment the anti-tumor agent is MK-663 (etoricoxib),see International Publication Number WO 1998/03484, the contents ofwhich are incorporated by reference in its entirety for all purposes.The structure for etoricoxib is shown below:

In one preferred embodiment the anti-tumor agent is COX-189(Lumiracoxib), see International Publication Number WO 1999/11605, thecontents of which are incorporated by reference in its entirety for allpurposes. The structure for Lumiracoxib is shown below:

In one preferred embodiment the anti-tumor agent is BMS-347070, see U.S.Pat. No. 6,180,651, the contents of which are incorporated by referencein its entirety for all purposes. The structure for BMS-347070 is shownbelow:

In one preferred embodiment the anti-tumor agent is NS-398 (CAS123653-11-2). The structure for NS-398 (CAS 123653-11-2) is shown below:

In one preferred embodiment the anti-tumor agent is RS 57067 (CAS17932-91-3). The structure for RS-57067 (CAS 17932-91-3) is shown below:

In one preferred embodiment the anti-tumor agent is4-Methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoyl-phenyl)-1H-pyrrole. Thestructure for4-Methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoyl-phenyl)-1H-pyrrole isshown below:

In one preferred embodiment the anti-tumor agent is2-(4-Ethoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl)-1H-pyrrole. Thestructure for2-(4-Ethoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl)-1H-pyrrole is shownbelow:

In one preferred embodiment the anti-tumor agent is meloxicam. Thestructure for meloxicam is shown below:

Other useful inhibitors as anti-tumor agents used in conjunction withantibodies of the present invention and pharmaceutical compositionsdescribed herein include aspirin, and non-steroidal anti-inflammatorydrugs (NSAIDs) which inhibit the enzyme that makes prostaglandins(cyclooxygenase I and II), resulting in lower levels of prostaglandins,include but are not limited to the following, Amigesic (salsalate),DOLOBID® (diflunisal), MOTRIN® (ibuprofen), ORUDIS® (ketoprofen),RELAFEN® (Nabumetone), FELDENE® (piroxicam), ALEVE® (naproxen),NAPROSYN® (naproxen), VOLTAREN® (diclofenac), INDOCIN® (indomethacin),CLINORIL® (sulindac), TOLECTIN® (tolmetin), LODINE® (etodolac), TORADOL®(ketorolac), DAYPRO® (oxaprozin) and combinations thereof. PreferredCOX-1 inhibitors include MOTRIN® (ibuprofen), NUPRIN® (ibuprofen),ALEVE® (naproxen), INDOCIN® (indomethacin), RELAFEN® (nabumetone), andcombinations thereof.

Targeted agents used in conjunction with an anti-ALK-1 antibody orantigen binding portion thereof, as described herein, and pharmaceuticalcompositions thereof as described herein, include EGFr inhibitors suchas IRESSA® (gefitinib, AstraZeneca), TARCEVA® (erlotinib or OSI-774, OSIPharmaceuticals Inc.), ERBITUX® ® (cetuximab, Imclone Pharmaceuticals,Inc.), EMD-7200 (Merck AG), ABX-EGF (Amgen Inc. and Abgenix Inc.), HR3(Cuban Government), IgA antibodies (University of Erlangen-Nuremberg),TP-38 (IVAX), EGFR fusion protein, EGF-vaccine, anti-EGFrimmunoliposomes (Hermes Biosciences Inc.) and combinations thereof.

Preferred EGFr inhibitors include IRESSA® (gefitinib), ERBITUX®(cetuximab), TARCEVA® (erlotinib) and combinations thereof.

The present invention also relates to anti-tumor agents selected frompan erb receptor inhibitors or ErbB2 receptor inhibitors, such asCP-724,714 (Pfizer, Inc.), CI-1033 (canertinib, Pfizer, Inc.),HERCEPTIN® (trastuzumab, Genentech Inc.), OMNITARG® (2C4, pertuzumab,Genentech Inc.), TAK-165 (Takeda), GW -572016 (Ionafarnib,GlaxoSmithKline), GW-282974 (GlaxoSmithKline), EKB-569 (Wyeth),PKI-166(Novartis), dHER2 (HER2 Vaccine, Corixa and GlaxoSmithKline), APC8024(HER2 Vaccine, Dendreon), anti-HER2/neu bispecific antibody (DecofCancer Center), B7.her2.IgG3 (Agensys), AS HER2 (Research Institute forRad Biology & Medicine), trifunctional bispecific antibodies (Universityof Munich) and mAB AR-209 (Aronex Pharmaceuticals Inc) and mAB 2B-1(Chiron) and combinations thereof. Preferred erb selective anti-tumoragents include HERCEPTIN® (trastuzumab), TAK-165, CP-724,714, ABX-EGF,HER3 and combinations thereof. Preferred pan erbb receptor inhibitorsinclude GW572016, CI-1033, EKB-569, and OMNITARG® (pertuzumab) andcombinations thereof.

Additional erbB2 inhibitors include those in WO 98/02434 (published Jan.22, 1998), WO 99/35146 (published Jul. 15, 1999), WO 99/35132 (publishedJul. 15, 1999), WO 98/02437 (published Jan. 22, 1998), WO 97/13760(published Apr. 17, 1997), WO 95/19970 (published Jul. 27, 1995), U.S.Pat. No. 5,587,458 (issued Dec. 24, 1996), and U.S. Pat. No. 5,877,305(issued Mar. 2, 1999), each of which is herein incorporated by referencein its entirety. For additional ErbB2 receptor inhibitors useful in thepresent invention, see U.S. Pat. Nos. 6,465,449, and 6,284,764, andInternational Application No. WO 2001/98277 each of which are hereinincorporated by reference in their entirety.

Additionally, other anti-tumor agents may be selected from the followingagents, Sorafenib (Onyx Pharmaceuticals Inc.; BAY-43-9006), GENASENSE®(augmerosen, Genta), Panitumumab (Abgenix/Amgen), ZEVALIN® (ibritumomabtiuxetan) (Schering), BEXXAR® (tositumomab) (Corixa/GlaxoSmithKline),Abarelix, ALIMTA® (pemetrexed), EPO 906 (Novartis), discodermolide(XM-296), ABT-510 (Abbott), NEOVASTAT® (Aeterna), enzastaurin (EliLilly), Combrestatin A4P (Oxigene), ZD-6126 (AstraZeneca), flavopiridol(Aventis), CYC-202 (Cyclacel), AVE-8062 (Aventis), DMXAA(Roche/Antisoma), Thymitaq (nolatrexed dihydrochloride, Eximias),TEMODAR® (temozolomide, Schering Plough) and REVLIMID® (lenalidomide)(Celegene) and combinations thereof.

Other anti-tumor agents may be selected from the following agents,CYPAT® (cyproterone acetate), Histerelin (histrelin acetate), PLENAXIS®(abarelix depot), Atrasentan (ABT-627), Satraplatin (JM-216),THALOMID®(thalidomide), THERATOPE® , tesmilifene (DPPE), ABI-007 (paclitaxel),EVISTA® (raloxifene), Atamestane (Biomed-777), XYOTAX® (polyglutamatepaclitaxel), TARGRETIN® (bexarotene) and combinations thereof.

Additionally, other anti-tumor agents may be selected from the followingagents, Trizaone (tirapazamine), APTOSYN® (exisulind), NEOVASTAT®(AE-941), CEPLENE® (histamine dihydrochloride), ORATHECIN® (rubitecan),VIRULIZIN®, Gastrimmune (G17DT), DX-8951f (exatecan mesylate), ONCONASE®(ranpirnase), BEC2 (mitumoab), XCYTRIN® (motexafin gadolinium) andcombinations thereof.

Further anti-tumor agents may be selected from the following agents,CeaVac (CEA), NEUTREXIN® (trimestrexate glucuronate) and combinationsthereof. Additional anti-tumor agents may selected from the followingagents, OVAREX® (oregovomab), OSIDEM® (IDM-1), and combinations thereof.Additional anti-tumor agents may be selected from the following agents,ADVEXIN® (ING 201), Tirazone (tirapazamine), and combinations thereof.Additional anti-tumor agents may selected from the following agents,RSR13 (efaproxiral), COTARA (131I chTNT 1/b), NBI-3001 (IL-4) andcombinations thereof. Additional anti-tumor agents may be selected fromthe following agents, CANVAXIN®, GMK vaccine, PEG Intron A, TAXOPREXIN®(DHA/paciltaxel) and combinations thereof. Other preferred anti-tumoragents include Pfizer's MEK½inhibitor PD325901, Array Biopharm's MEKinhibitor ARRY-142886, Bristol Myers' CDK2 inhibitor BMS-387,032,Pfizer's CDK inhibitor PD0332991 and AstraZeneca's AXD-5438 andcombinations thereof. Additionally, mTOR inhibitors may also be utilizedsuch as CCI-779 (Wyeth) and rapamycin derivatives RAD001 (Novartis) andAP-23573 (Ariad), HDAC inhibitors SAHA (Merck Inc./Aton Pharmaceuticals)and combinations thereof. Additional anti-tumor agents include aurora 2inhibitor VX-680 (Vertex), Chk½inhibitor XL844 (Exilixis).

The following cytotoxic agents, e.g., one or more selected from thegroup consisting of ELLENCE® (epirubicin), TAXOTERE® (docetaxel)paclitaxel, ZINECARD® (dexrazoxane), RITUXIN® (rituximab), GLEEVEC®(imatinib mesylate), and combinations thereof, may be used inconjunction with a Anti-ALK-1 antibody or antigen binding portionthereof, as described herein, and pharmaceutical compositions thereof,as described herein.

The invention also contemplates the use of the antibodies andantigen-binding portions thereof of the present invention together withhormonal therapy, including but not limited to, AROMASIN® (exemestane,Pfizer Inc.), LUPRON® or Leuplin (leuprorelin, TAP PharmaceuticalProducts Inc./Abbott/Takeda), ARIMIDEX® (anastrozole, Astrazeneca),ZOLADEX® (goserelin, AstraZeneca), doxercalciferol, fadrozole,formestane, tamoxifen citrate NOLVADEX® (tamoxifen, tamoxien citrate,AstraZeneca), CASODEX® (bicalutamide, AstraZeneca), Abarelix (PraecisPharmaceuticals), TRELSTAR® (triptorelin pamoate), and combinationsthereof.

The invention also relates to hormonal therapy agents such asanti-estrogens including, but not limited to fulvestrant, toremifene,raloxifene, lasofoxifene, FERMARA® (letrozole, Novartis), anti-androgenssuch as bicalutamide, flutamide, mifepristone, nilutamide, CASODEX®(4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3′-(trifluoromethyl)propionanilide,bicalutamide) and combinations thereof.

Further, the invention provides antibodies of the present inventionalone or in combination with one or more supportive care products, e.g.,a product selected from the group consisting of Filgrastim (Neupogen)ZOFRAN® (ondansetron), FRAGMIN® (dalteparin), PROCRIT® (epoetin alfa),ALOXI® (palonosetron hydrochloride), EMEND® (aprepitant), orcombinations thereof.

Particularly preferred cytotoxic agents include CAMPTOSAR® (irinotecanHCI), ERBITUX® (cetuximab, IRESSA® (gefitinib), GLEEVEC® (imatinibmesylate), TAXOTERE® (docetaxel), and combinations thereof.

The following topoisomerase I inhibitors may be utilized as anti-tumoragents camptothecin, CAMPTOSAR® (irinotecan HCl, edotecarin, ORATHECIN®(SuperGen), exatecan (Daiichi), BN-80915 (Roche) and combinationsthereof. Particularly preferred toposimerase II inhibitors includeELLENCE® (epirubicin).

The antibodies of the invention may be used with antitumor agents,alkylating agents, antimetabolites, antibiotics, plant-derived antitumoragents, camptothecin derivatives, tyrosine kinase inhibitors, otherantibodies, interferons, and/or biological response modifiers.

Alkylating agents include, but are not limited to, nitrogen mustardN-oxide, cyclophosphamide,ifosfamide, melphalan, busulfan, mitobronitol,carboquone, thiotepa, ranimustine, nimustine,temozolomide, AMD-473,altretamine, AP-5280, apaziquone, brostallicin, bendamustine,carmustine, estramustine, fotemustine, glufosfamide, ifosfamide,KW-2170, mafosfamide, and mitolactol; platinum-coordinated alkylatingcompounds include but are not limited to, cisplatin, PARAPLATIN®(carboplatin), eptaplatin, lobaplatin, nedaplatin, ELOXATIN®(oxaliplatin, Sanofi) or satrplatin and combinations thereof.Particularly preferred alkylating agents include ELOXATIN®(oxaliplatin).

Antimetabolites include but are not limited to, methotrexate,6-mercaptopurine riboside,mercaptopurine, 5-fluorouracil (5-FU) alone orin combination with leucovorin, tegafur, UFT, doxifluridine,carmofur,cytarabine, cytarabine ocfosfate, enocitabine, S-1, ALIMTA® (pemetrexeddisodium, LY231514, MTA), GEZMAR® (gemcitabine, Eli Lilly), fludarabin,5-azacitidine, capecitabine, cladribine, clofarabine,decitabine,eflornithine, ethynylcytidine, cytosine arabinoside, hydroxyurea, TS-1,melphalan, nelarabine, nolatrexed, ocfosfate, pemetrexed disodium,pentostatin, pelitrexol, raltitrexed, triapine, trimetrexate,vidarabine, vincristine, vinorelbine; or for example, one of thepreferred anti-metabolites disclosed in European Patent Application No.239362 such asN-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamicacid and combinations thereof.

Antibiotics include intercalating antibiotics but are not limited to:aclarubicin, actinomycin D, amrubicin, annamycin, adriamycin, bleomycin,daunorubicin, doxorubicin, elsamitrucin, epirubicin, galarubicin,idarubicin, mitomycin C, nemorubicin, neocarzinostatin, peplomycin,pirarubicin, rebeccamycin, stimalamer, streptozocin, valrubicin,zinostatin and combinations thereof.

Plant derived anti-tumor substances include for example those selectedfrom mitotic inhibitors, for example vinblastine, TAXOTERE® (docetaxel),paclitaxel and combinations thereof.

Cytotoxic topoisomerase inhibiting agents include one or more agentsselected from the group consisting of aclarubicn, amonafide, belotecan,camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan,(CAMPTOSAR® ) (irinotecan HCl), edotecarin, ELLENCE® (epirubicin),etoposide, exatecan, gimatecan, lurtotecan, mitoxantrone, pirarubicin,pixantrone, rubitecan, sobuzoxane, SN-38, tafluposide, topotecan, andcombinations thereof.

Preferred cytotoxic topoisomerase inhibiting agents include one or moreagents selected from the group consisting of camptothecin,10-hydroxycamptothecin, 9-aminocamptothecin, CAMPTOSAR® (irinotecanHCl), edotecarin, ELLENCE® (epirubicin), etoposide, SN-38, topotecan,and combinations thereof.

Immunologicals include interferons and numerous other immune enhancingagents. Interferons include interferon alpha, interferon alpha-2a,interferon, alpha-2b interferon beta, interferon gamma-1a, interferongamma-1b (ACTIMMUNE®), or interferon gamma-n1 and combinations thereof.Other agents include filgrastim, lentinan, sizofilan, THERACYS®,ubenimex, WF-10, aldesleukin, alemtuzumab, BAM-002, dacarbazine,daclizumab, denileukin, gemtuzumab ozogamicin, ibritumomab, imiquimod,lenograstim, lentinan, melanoma vaccine (Corixa Corporation),molgramostim, OncoVAX-CL, sargramostim, tasonermin, tecleukin,thymalasin, tositumomab, VIRULIZIN®, Z-100, epratuzumab, mitumomab,oregovomab, pemtumomab (Y-muHMFG1), PROVENGE® (Dendreon Corp.) andcombinations thereof.

Biological response modifiers are agents that modify defense mechanismsof living organisms or biological responses, such as survival, growth,or differentiation of tissue cells to direct them to have anti-tumoractivity. Such agents include krestin, lentinan, sizofuran, picibanil,ubenimex and combinations thereof.

Other anticancer agents include alitretinoin, ampligen, atrasentanbexarotene, bortezomib. Bosentan, calcitriol, exisulind, finasteride,fotemustine, ibandronic acid, miltefosine, mitoxantrone, I-asparaginase,procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin,tazarotne, TELCYTA® (TLK-286, Telik Inc.), VELCADE® (bortemazib,Millenium), tretinoin, and combinations thereof.

Other anti-angiogenic compounds include acitretin, fenretinide,thalidomide, zoledronic acid,angiostatin, aplidine, cilengtide,combretastatin A-4, endostatin, halofuginone, rebimastat, removab,REVLIMID® (lenalidomide),squalamine, ukrain, VITAXIN® and combinationsthereof.

Platinum-coordinated compounds include but are not limited to,cisplatin, carboplatin, nedaplatin, oxaliplatin, and combinationsthereof.

Camptothecin derivatives include but are not limited to camptothecin,10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan, SN-38,edotecarin, topotecan and combinations thereof.

Other antitumor agents include mitoxantrone, 1-asparaginase,procarbazine, dacarbazine, hydroxycarbamide, pentostatin, tretinoin andcombinations thereof.

Anti-tumor agents capable of enhancing antitumor immune responses, suchas CTLA-4 (cytotoxic lymphocyte antigen 4) antibodies, and other agentscapable of blocking CTLA-4 may also be utilized, such as MDX-010(Medarex) and CTLA-4 compounds disclosed in U.S. Pat. No. 6,682,736; andanti-proliferative agents such as other farnesyl protein transferaseinhibitors, for example the farnesyl protein transferase inhibitors. Foradditional, specific CTLA-4 antibodies that can be used in the presentinvention see U.S. Provisional Application 60/113,647 (filed Dec. 23,1998), U.S. Pat. No. 6,682,736 both of which are herein incorporated byreference in their entirety. For example, another anti-CTLA-4 antibodythat can be used in accordance with the present invention isticilimumab, which has the sequence of monoclonal antibody 11.2.1 inU.S. Pat. No. 6,682,736.

For specific IGF1R antibodies that can be used in the present invention,see International Patent Application No. WO 2002/053596, which is hereinincorporated by reference in its entirety.

For specific CD40 antibodies that can be used in the present invention,see International Patent Application No. WO 2003/040170, which is hereinincorporated by reference in its entirety.

Gene therapy agents may also be employed as anti-tumor agents such asTNFerade (GeneVec), which express TNFalpha in response to radiotherapy.

In one embodiment of the present invention, statins may be used inconjunction with a Anti-ALK-1 antibody or antigen binding portionthereof, as described herein, and pharmaceutical compositions thereof.Statins (HMG-CoA reducatase inhibitors) may be selected from the groupconsisting of LIPITOR® (atorvastatin Pfizer Inc.), PRAVACHOL®(pravastatin, Bristol-Myers Squibb), MEVACOR® (lovastatin, Merck Inc.),ZOCOR® (simvastatin, Merck Inc.), LESCOL® (fluvastatin, Novartis),BAYCOL® (cerivastatin,Bayer), CRESTOR® (rosuvastatin, AstraZeneca),ADVICOR® (lovostatin and niacin, Kos Pharmaceuticals), derivatives andcombinations thereof.

In a preferred embodiment the statin is selected from the groupconsisting of Atovorstatin and Lovastatin, derivatives and combinationsthereof.

Other agents useful as anti-tumor agents include CADUET® (amlodipinebesylate and atorvastatin).

For any of the methods of treating a hyperproliferative disorder orabnormal cell growth as described herein using a combination of ananti-ALK-1 antibody or antigen binding portion with at least oneadditional therapeutic agent, the anti-ALK-1 antibody can be conjugated,or derivatized, with the additional therapeutic agent. The at least oneadditional therapeutic agent can also be administered separately, or ina non-derivatized or non-conjugated manner. When the at least oneadditional therapeutic agent is not derivatized or conjugated to theantibody, it can be administered within the same pharmaceuticalformulation as the antibody, or it can be administered in a separateformulation.

Treatment of Vision Loss

The inventive compounds and pharmaceutical compositions containing them,are useful for treating severe vision loss from age-related maculardegeneration and other diseases affecting the posterior segment of theeye, such as choroidal neovascularization, diabetic retinopathy,glaucoma, retinitis pigmentosa, and the like.

For example, the inventive compositions may be used to form a drug depotbehind the eye and may include one or more pharmaceutically activeagents, in addition to one or more non-active excipients as describedherein. Examples of pharmaceutically active agents useful in theinventive compositions includes anti-infectives, including, withoutlimitation, antibiotics, antivirals, and antifungals; antiallergenicagents and mast cell stabilizers; steroidal and nonsteroidalanti-inflammatory agents (such as nepafenac); cyclooxygenase inhibitors,including, without limitation, Cox I and Cox II inhibitors; combinationsof anti-infective and anti-inflammatory agents; decongestants;anti-glaucoma agents, including, without limitation, adrenergics,beta-adrenergic blocking agents, alpha-adrenergic agonists,parasypathomimetic agents, cholinesterase inhibitors, carbonic anhydraseinhibitors, and prostaglandins; combinations of anti-glaucoma agents;antioxidants; nutritional supplements; drugs for the treatment ofcystoid macular edema including, without limitation, non-steroidalanti-inflammatory agents; drugs for the treatment of age related maculardegeneration (AMD) including nonexudative (dry) and exudative (wet) AMD,including, without limitation, angiogenesis inhibitors, includingangiogenesis inhibitors that inhibit protein kinase receptors, includingprotein kinase receptors that are VEGF receptors; and nutritionalsupplements; drugs for the treatment of herpetic infections and CMVocular infections; drugs for the treatment of proliferativevitreoretinopathy including, without limitation, antimetabolites andfibrinolytics; wound modulating agents, including, without limitation,growth factors; antimetabolites; neuroprotective drugs, including,without limitation, eliprodil; and angiostatic steroids for thetreatment of diseases or conditions of posterior segment 26, including,without limitation, age related macular degeneration (AMD) includingnonexudative (dry) and exudative (wet) AMD, choroidalneovascularization, retinopathies, retinitis, uveitis, macular edema,and glaucoma. For additional information about such angiostatic steroidssee U.S. Pat. Nos. 5,679,666 and 5,770,592. A non-steroidalanti-inflammatory for the treatment of cystoid macular edema isnepafenac.

For administration to the eye, a compound of the present invention isdelivered in a pharmaceutically acceptable ophthalmic vehicle such thatthe compound is maintained in contact with the ocular surface for asufficient time period to allow the compound to penetrate the corneaand/or sclera and internal regions of the eye, including, for example,the anterior chamber, posterior chamber, vitreous body, aqueous humor,vitreous humor, cornea, iris/ciliary's, lens, choroid/retina and sclera.The pharmaceutically acceptable ophthalmic vehicle may be an ointment,vegetable oil, or an encapsulating material. A compound of the inventionmay also be injected directly into the vitreous humor or aqueous humor.

Further, a compound may be also be administered by well known,acceptable methods, such as sub-Tenon and/or subconjunctival injections.As is well known in the ophthalmic art, the macula is comprisedprimarily of retinal cones and is the region of maximum visual acuity inthe retina. A Tenon's capsule or Tenon's membrane is disposed on thesclera. A conjunctiva covers a short area of the globe of theeyeposterior to the limbus (the bulbar conjunctiva) and folds up (theupper cul-de-sac) or down (the lower cul-de-sac) to cover the innerareas of the upper eyelid and lower eyelid, respectively. Theconjunctiva is disposed on top of Tenon's capsule. The sclera andTenon's capsule define the exterior surface of the globe of the eye. Fortreatment of ocular diseases such as age related macular degeneration(AMD) including nonexudative (dry) and exudative (wet) AMD, choroidalneovascularization, retinopathies (such as diabetic retinopathy,retinopathy of prematurity), diabetic macular edema, retinitis, uveitis,cystoid macular edema (CME), glaucoma, and other diseases or conditionsof the posterior segment of the eye, it is preferable to dispose a depotof a specific quantity of an ophthalmically acceptable pharmaceuticallyactive agent directly on the outer surface of the sclera and belowTenon's capsule. In addition, in cases of age related maculardegeneration (AMD) including nonexudative (dry) and exudative (wet) AMDand CME it is most preferable to dispose the depot directly on the outersurface of the sclera, below Tenon's capsule, and generally above themacula.

The compounds may be formulated as a depot preparation. Such long-actingformulations may be administered by implantation (for example,subcutaneously or intramuscularly) intramuscular injection or by theabove mentioned sub-Tenon or intravitreal injection. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Within particularly preferred embodiments of the invention, thecompounds may be prepared for topical administration in saline (combinedwith any of the preservatives and antimicrobial agents commonly used inocular preparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, the present compositions, prepared asdescribed above, may also be administered directly to the cornea.

Within preferred embodiments, the composition is prepared with amuco-adhesive polymer that binds to cornea. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) orion-exchange resins, or as sparingly soluble derivatives, for example,as a sparingly soluble salt.

A pharmaceutical carrier for hydrophobic compounds is a cosolvent systemcomprising benzyl alcohol, a nonpolar surfactant, a water-miscibleorganic polymer, and an aqueous phase. The cosolvent system may be a VPDco-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol300, made up to volume in absolute ethanol. The VPD co-solvent system(VPD:5W) contains VPD diluted 1:1 with a 5% dextrose in water solution.This co-solvent system dissolves hydrophobic compounds well, and itselfproduces low toxicity upon systemic administration. Naturally, theproportions of a co-solvent system may be varied considerably withoutdestroying its solubility and toxicity characteristics. Furthermore, theidentity of the co-solvent components may be varied: for example, otherlow-toxicity nonpolar surfactants may be used instead of polysorbate 80;the fraction size of polyethylene glycol may be varied; otherbiocompatible polymers may replace polyethylene glycol, e.g. polyvinylpyrrolidone; and other sugars or polysaccharides may be substituted fordextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are known examples ofdelivery vehicles or carriers for hydrophobic drugs. Certain organicsolvents such as dimethylsulfoxide also may be employed, althoughusually at the cost of greater toxicity. Additionally, the compounds maybe delivered using a sustained-release system, such as semipermeablematrices of solid hydrophobic polymers containing the therapeutic agent.Various sustained-release materials have been established and are knownby those skilled in the art. Sustained-release capsules may, dependingon their chemical nature, release the compounds for a few weeks up toover 100 days. Depending on the chemical nature and the biologicalstability of the therapeutic reagent, additional strategies for proteinstabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid- orgel-phase carriers or excipients. Examples of such carriers orexcipients include calcium carbonate, calcium phosphate, sugars,starches, cellulose derivatives, gelatin, and polymers such aspolyethylene glycols.

Any of the compositions can be formulated for administration to anindividual. An individual of the present invention is preferably amammal, or more preferably a human.

The pharmaceutical formulations herein can further include a therapeuticagent selected from the group consisting of: an antineoplastic agent, ananti-inflammatory agent, an antibacterial agent, an antiviral agent, anangiogenic agent, and an anti-angiogenic agent. Examples of such agentsare disclosed herein.

For example, an antineoplastic agent may be selected from the groupconsisting of Acodazole Hydrochloride; Acronine; Adozelesin;Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate;Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase;Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa;Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; DaunorubicinHydrochloride; Decitabine; Dexormaplatin; Dezaguanine; DezaguanineMesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin;Edatrexate; Eflornithine Hydrochloride Elsamitrucin; Enloplatin;Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole;Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate;Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine;Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone;Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198;Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Imofosine; InterferonAlfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3;Interferon Beta-1a; Interferon Gamma-1b; Iproplatin; IrinotecanHydrochloride; Lanreotide Acetate; Letrozole; Leuprolide AcetateLiarozole Hydrochloride; Lometrexol Sodium; Lomustine; LosoxantroneHydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride;Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril;Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine;Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin;Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; MycophenolicAcid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel;Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycinl, SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid;Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin;Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine;Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; ToremifeneCitrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate;Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; UracilMustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate;Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate;Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate;Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin;Zinostatin; Zorubicin Hydrochloride.

An anti-angiogenic agents are any agents that inhibit angiogenesis,whether disclosed herein or known in the art. In preferred embodiments,an anti-angiogenic agent is an anti-VEGF agent, such as MACUGEN®(pegaptanib sodium) (Eyetech, New York, N.Y.); or anti-VEGF antibody.

Pharmaceutical compositions can be formulated by standard techniquesusing one or more suitable carriers, excipients, and dilutents. See,e.g., Remington's Pharmaceutical Sciences, (19^(th) Ed. Williams &Wilkins, 1995) (incorporated herein by reference for all purposes).

Formulations suitable for parenteral administration include aqueous andnon-aqueous formulations isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending systems designed to target the compound to bloodcomponents or one or more organs. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules orvials. For intraocular formulations, unit dosages are preferred becauseno preservatives are in the formulation. For other parenteralformulations, preservative may be used, which would allow for multi dosecontainers

Extemporaneous injections solutions and suspensions may be prepared, forexample, from sterile powders. Parenteral and intravenous forms may alsoinclude minerals and other materials to make them compatible with thetype of injection or delivery system chosen.

Particular parenteral administrations contemplated by the presentinvention include intraocular and intravitreous administrations to theeye. Pharmaceutical formulations for intraocular and intravitreousadministrations include phosphate buffered saline (PBS) and balancedisotonic salt solution (BSS) with or without excipients such as mannitolor sorbitol as protein stabilizers.

In general, water, suitable oil, saline, aqueous dextrose (glucose), orrelated sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration preferably contain a watersoluble salt of the active ingredient, suitable stabilizing agents and,if necessary, buffer substances. Antioxidizing agents, such as sodiumbisulfite, sodium sulfite, or ascorbic acid, either alone or combined,are suitable stabilizing agents. Also used are citric acid saltsthereof, or sodium EDTA. In addition, parenteral solutions may containpreservatives, such as benzalkonium chloride, methyl- or propyl-paraben,or chlorobutanol. Suitable pharmaceutical carriers are described inRemington, cited supra.

In any of the embodiments herein, a composition or pharmaceuticalformulation herein may by lypholized.

In any of the embodiments herein, the pharmaceutical formulationspreferable have less than about 10, more preferably less than about 5,more preferably less than about 3, or more preferably less than about 1endotoxin unit(s) per milligram of therapeutic agents.

In some embodiments, the methods of treatment disclosed herein furtherinclude administering to an individ ual suffering from an angiogeniccondition one or more therapeutic agents selected from the groupconsisting of antineoplastic agents, antiviral agents, anti-inflammatoryagents, antibacterial agents, anti-angiogenic agents, or anti-angiogenicagents.

Such combination treatments can be achieved by either administering toan individual a co -formulating of the compositions herein with theadditional therapeutic agent(s) or by administering the compositionsherein and the therapeutic agent(s) as two separate pharmaceuticalformulations. In embodiments wherein more than onecomposition/therapeutic agent is administered to an individual, lowerdosages of the compositions and/or therapeutic agent(s) may be utilizedas a result of the synergistic effect of both active ingredients.

Antineoplastic agents that may be administered to an individual include,but are not limited to, Aclarubicin; Acodazole Hydrochloride; Acronine;Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate;Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase;Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa;Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; DaunorubicinHydrochloride; Decitabine; Dexormaplatin; Dezaguanine; DezaguanineMesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin;Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin;Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole;Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate;Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine;Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone;Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198;Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Imofosine; InterferonAlfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3;Interferon Beta-1a; Interferon Gamma-1b; Iproplatin; IrinotecanHydrochloride; Lanreotide Acetate; Letrozole; Leuprolide AcetateLiarozole Hydrochloride; Lometrexol Sodium; Lomustine; LosoxantroneHydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride;Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril;Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine;Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin;Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; MycophenolicAcid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel;Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycinl, SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid;Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin;Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine;Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; ToremifeneCitrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate;Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; UracilMustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate;Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate;Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate;Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin;Zinostatin; Zorubicin Hydrochloride.

Antibacterial agents that may be administered to an individual include,but are not limited to, penicillins, aminoglycosides, macrolides,monobactams, rifamycins, tetracyclines, chloramphenicol, clindamycin,lincomycin, imipenem, fusidic acid, novobiocin, fosfomycin, fusidatesodium, neomycin, polymyxin, capreomycin, colistimethate, colistin,gramicidin, minocycline, doxycycline, vanomycin, bacitracin, kanamycin,gentamycin, erythromicin and cephalosporins.

Anti-inflammatory agents that may be administered to an individualinclude, but are not limited to, NSAIDS (e.g., aspirin (salicylamide),sodium salicylamide, indoprofen, indomethacin, sodium indomethacintrihydrate, BAYER®, BUFFERIN®, CELEBREX®, (celecoxib), diclofenac,ECOTRIN® (aspirin), diflunisal, fenoprofen, naproxen, sulindac, VIOXX®(rofecoxib), corticosteroids or corticotropin (ACTH), colchicine, andanecortave acetate.

Antiviral agents that may be administered to an individual include, butare not limited to, α-methyl-P-adamantane methylamine,1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide,9-[2-hydroxy-ethoxy]methylguanine, adamantanamine,5-iodo-2′-deoxyuridine, trifluorothymidine, interferon, adeninearabinoside, CD4,3′-azido-3′-deoxythymidine (AZT),9-(2-hydroxyethoxymethyl)-guanine (acyclovir), phosphonoformic acid,1-adamantanamine, peptide T, and 2′,3′dideoxycytidine.

Administration of a composition of the present invention to a targetcell in vivo can be accomplished using any of a variety of techniqueswell known to those skilled in the art.

For example, compositions of the present invention can be administeredsystemically or locally by any means known in the art (e.g., orally,intraocularly, intravascularly (i.v.), intradermally, intramuscularly,transdermally, transmucosally, enterically, parentally, by inhalationspray, rectally, or topically) in dosage unit formulations andcontaining conventional pharmaceutically acceptable carriers, adjuvants,and vehicles.

As used herein the term intraocularly includes intravitreal,sub-retinal, and the like.

As used herein the term parenteral as used herein includes,subcutaneous, intravenous, intramuscular, intrasternal, infusiontechniques or intraperitoneally. Suppositories for rectal administrationof the drug can be prepared by mixing the drug with a suitablenon-irritating excipient such as cocoa butter and polyethylene glycolsthat are solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum and release the drug.

The dosage regimen for treating a disorder or a disease with thecompositions of this invention is based on a variety of factors,including the type of disease, the age, weight, sex, medical conditionof the patient, the severity of the condition, the route ofadministration, and the particular compound employed. Thus, the dosageregimen can vary widely, but can be determined routinely using standardmethods.

For systemic administration, the anti-ALK-1 antibody or antigen-bindingportion thereof of the present invention and/or one or more additionaltherapeutic agents are preferably administered at a dose of at least0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0,5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 75, 100, or 150 mg/kg bodyweight. In other embodiments, the polypeptides (preferably dimers orhomodimers) and/or small molecules herein are administered systemicallyat a dose of 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, morepreferably 1-30 mg/kg body weight, or more preferably 5-20 mg/kg.

For localized administration, the anti-ALK-1 antibody or antigen-bindingportion thereof of the present invention and/or one or more additionaltherapeutic agents are preferably administered at a dose of at least 50μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500μg, 550 μg, 600 μg, 650 μg, or 700 μg. In other embodiments, thepolypeptides (preferably dimers or homodimers) and/or small moleculesherein are administered locally at a dose of 50-1000 μg, more preferably100-800 μg, more preferably 200-500 μg, or more preferably 300-400 μgper site.

For example, for dermal administration the anti-ALK-1 antibody orantigen-binding portion thereof of the present invention and/orpeptidomimetics and/or one or more additional therapeutic agents areadministered at a dose of 50-1000 μg/cm², more preferably 100-800μg/cm², or more preferably 200-500 μg/cm². In another example, forocular administration, the polypeptides and/or peptidomimetics and/orsmall molecules of the present invention are administered at a dose of50-1000 μg/eye, more preferably 100-800 μg/eye, or more preferably200-500 μg/eye.

The pharmaceutical compositions preferably include the active ingredient(e.g., an anti-ALK-1 antibody) in an effective amount, i.e., in anamount effective to achieve therapeutic or prophylactic benefit. Theactual amount effective for a particular application will depend on thecondition being treated and the route of administration. Determinationof an effective amount is well within the capabilities of those skilledin the art, especially in light of the disclosure herein.

Preferably, the effective amount of the active ingredient, e.g., ananti-ALK-1 antibody, is from about 0.0001 mg to about 500 mg activeagent per kilogram body weight of a patient, more preferably from about0.001 to about 250 mg active agent per kilogram body weight of thepatient, still more preferably from about 0.01 mg to about 100 mg activeagent per kilogram body weight of the patient, yet still more preferablyfrom about 0.5 mg to about 50 mg active agent per kilogram body weightof the patient, and most preferably from about 1 mg to about 15 mgactive agent per kilogram body weight of the patient.

In terms of weight percentage, the formulations of the present inventionwill preferably comprise the active agent, e.g., an anti-ALK-1 antibody,in an amount of from about 0.0001 to about 10 wt. %, more preferablyfrom about 0.001 to about 1 wt. %, more preferably from about 0.05 toabout 1 wt. %, or more preferably about 0.1 wt. to about 0.5 wt. %.

Gene Therapy

The nucleic acid molecules that encode the antibodies and antibodyportions of the present invention can be administered to a patient inneed thereof via gene therapy. The therapy may be either in vivo or exvivo. In a preferred embodiment, nucleic acid molecules encoding both aheavy chain and a light chain are administered to a patient. In a morepreferred embodiment, the nucleic acid molecules are administered suchthat they are stably integrated into chromosomes of B cells becausethese cells are specialized for producing antibodies. In a preferredembodiment, precursor B cells are transfected or infected ex vivo andre-transplanted into a patient in need thereof. In another embodiment,precursor B cells or other cells are infected in vivo using a virusknown to infect the cell type of interest. Typical vectors used for genetherapy include liposomes, plasmids, and viral vectors. Exemplary viralvectors are retroviruses, adenoviruses and adeno-associated viruses.After infection either in vivo or ex vivo, levels of antibody expressioncan be monitored by taking a sample from the treated patient and usingany immunoassay known in the art or discussed herein.

In a preferred embodiment, the gene therapy method comprises the stepsof administering an isolated nucleic acid molecule encoding the heavychain or an antigen-binding portion thereof of an anti-ALK-1 antibodyand expressing the nucleic acid molecule. In another embodiment, thegene therapy method comprises the steps of administering an isolatednucleic acid molecule encoding the light chain or an antigen-bindingportion thereof of an anti-ALK-1 antibody and expressing the nucleicacid molecule. In a more preferred method, the gene therapy methodcomprises the steps of administering an isolated nucleic acid moleculeencoding the heavy chain or an antigen-binding portion thereof and anisolated nucleic acid molecule encoding the light chain or theantigen-binding portion thereof of an anti-ALK-1 antibody of theinvention and expressing the nucleic acid molecules. The gene therapymethod may also comprise the step of administering another therapeuticagent, such as any of the agents discussed previously in connection withcombination therapy.

Method for Screening ALK-1 Antagonists or Agonists

In one embodiment, the present invention provides a method fordetermining if a substance inhibits up-regulation of a specificdownstream target gene of ALK-1, Id1, such as, for example, the TAQMAN®Assay for Id1 described in Example 12. The method comprises contacting asample of cells that express Id1 with the substance and determining ifId1 expression is inhibited, wherein a reduced level of Id1 expressionin the sample of cells contacted with the substance as compared to acontrol sample of cells is indicative of said substance inhibiting Id1expression. In one specific embodiment, the substance is an antibodythat binds to the extracellular domain of ALK-1. In another embodiment,the substance is a small molecule. According to the invention, the cellscan inherently express both ALK-1 and Id1, such as HUVECs described inExample 12, or which have been transformed or transfected with DNAencoding one or both of these. One can determine the expression of Id1via, e.g., the use of TAQMAN® Assay for Id1 described in Example 12.

Conversely, activators or agonists can also be tested for, or utilized,following the same type of procedures.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLES

In the following examples and preparations, “MW” means molecular weight;“His-Tag” means C-terminal polyhistidine (6×His) tag for rapidpurification with nickel-chelating resin and detection with an anti-His(C-term) antibody; “BSA” means bovine serum albumin; “EDTA” meansethylenediaminetetraacetic acid; “DMSO” means dimethyl sulfoxide; “MOPS”means 3-(N-morpholino) propanesulfonic acid; “MES” means2-(N-Morpholino)ethanesulfonic acid; “PBS” means phosphate bufferedsaline; “dPBS” means Dulbecco's phosphate buffered saline; “HEMA” means2-hydroxy-ethyl methacrylate; “DMEM” means Dulbecco's modified eagle'smedium; “FBS” means fetal bovine serum; “NEAA” means non-essential aminoacids; “HEPES” means N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid; and “DMF” means dimethyl formamide.

Example 1 ALK-1 Immunogen Preparation

The ECD of ALK-1 was cloned from full-length human ALK-1 ORF clone(Invitrogen, Clone ID IOH21048) by PCR using the forward5′-ACGGCCCAGCCGGCCGACCCTGTGAAGCCGTCT (SEQ ID NO: 96) and

reverse 5′-ACTAAGCTTTTAATGATGATGATGATGATGCTGGCCATCTGTTCCCG (SEQ ID NO:97)primers. The PCR product was purified, treated with the SfiI andHindIII restriction enzymes, and cloned into the SfiI/HindIII site of amammalian expression vector pSecTag2/Hygro (Invitrogen Inc., Catalog No.V910-20). The clone was used to transiently transfect 293T cells withFugene 6 transfection reagent (Roche Applied Science, Catalog No.1814443) following manufacture's instruction. Supernatant from the cellculture containing the secreted target protein was harvested 72 hourspost-transfection and allowed to bind to Ni-NTA resin (Qiagen, CatalogNo. 30430) at 4° C. overnight. The resin was then washed with buffercontaining 20 mM Tris pH 8.0, 25 mM imidazole and 300 mM sodiumchloride. The His-Tag protein was eluted off the resin using buffercontaining 20 mM Tris pH 8.0, 300 mM imidazole and 300 mM sodiumchloride. A CM Sepharose cation exchange resin was used to furtherpurify the protein in 20 mM sodium phosphate (pH 7.0), and the unboundfraction containing the target protein was collected. The protein wasbuffer exchanged to PBS or 10 mM HEPES, pH 7.4, plus 150 mM sodiumchloride by dialysis and concentrated to 0.2-1 mg/mL with a final purityof >90%, judged by SDS PAGE gel stained with Coomassie blue. The ALK-1ECD His-Tag protein was heavily glycosylated with an apparent MW of 26KDa, comparing to an 11 KDa theoretical MW for the protein. The ALK-1ECD His-Tag protein (SEQ ID NO: 98) has been used for generation ofhybridomas producing anti-ALK-1 antibody as described in Example 2.Human ALK-1 ECD His-Tag Protein:gene sequence (lowercase part is the secretion signal):

(SEQ ID NO: 99) atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacgcggcccagccggccGACCCTGTGAAGCCGTCTCGGGGCCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGGGCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCTGCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCACTACTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGCCACCCAACCTCCTTCGGAGCAGCCGGGAACAGATGGCCAGCATCATCATCATCATCATprotein sequence:

(SEQ ID NO: 98) DPVKPSRGPLVTCTCESPHCKGPTCRGAWCTVVLVREEGRHPQEHRGCGNLHRELCRGRPTEFVNHYCCDSHLCNHNVSLVLEATQPPSEQPGTDGQHHH HHH

Example 2 Generation of Hybridomas Producing Anti-ALK-1 Antibody

Eight to ten week old XENOMOUSE® mice were immunized in their hindfootpads with 10 μg/mouse of either recombinant human ALK-1/Fc chimera(R&D Systems, Inc., Catalog Number 370-AL) or with the ALK-1 ECD His-Tagprotein described in Example 1. This dose was repeated five to seventimes over a three to five week period. Three or four days beforefusion, the mice were given a final injection of the immunogen in PBS.The lymph node lymphocytes from immunized mice were fused with thenon-secretory myeloma P3-X63-Ag8.653 cell line via electro cell fusion(ATCC Cat. No. CRL 1580), and these fused cells were subjected toHA-DMEM selection as previously described (DMEM/15% FBS/1% 200 mML-glutamine/1% 100× Non-essential amino acid/1% 100× Pen/Strep/10 U/mlIL-6/1 vial/liter OPI media supplement plus 0.5×HA(Azaserine-Hypoxanthine, Sigma, Cat. # A9666)). A panel of hybridomaswas recovered that all secrete ALK-1 specific human IgG2 antibodies.

ELISA assay was used to detect antibody binding. Immunogen was coated tothe 96-well Immulon microtiter plate (NUNC-Immuno™ plate MaxiSorp™surface, Nalge Nunc International, Cat. No. 439454) at 4 μg/mL in 50 mMsodium bicarbonate buffer for overnight at 4° C. Plates were washed, andthen blocked with PBS with the addition of 0.1% Tween-20 and 0.5% bovineserum albumin. Antibodies were added to the blocked ELISA plates,incubated for 1 hour, and washed with PBS with Tween-20. The binding wasdetected by anti-human IgG-horseradish peroxidase (Pierce, Catalog No.31420) followed by the addition of ABTS (Pierce, Catalog No. 37615).Colorimetric measurements were performed at 405 nm in a micro-platereader (SpectraMax Plus 384, Molecular Devices).

Twenty five hybridomas were selected for further study. These weresingle-cell cloned by limiting dilution and were designated 1.11.1;1.12.1; 1.12.1(rWT); 1.12.1(M29I/D19A); 1.12.1(M29I); 1.12.1(D19A);1.13.1; 1.14.1; 1.151.1; 1.162.1; 1.183.1; 1.27.1; 1.29.1; 1.31.1;1.8.1; 1.9.1; 4.10.1; 4.24.1; 4.38.1; 4.58.1; 4.62.1; 4.68.1; 4.72.1;5.13.1; 5.34.1; 5.53.1; 5.56.1; 5.57.1; and 5.59.1.

Mouse Hybridoma Cell line LN 15916 (the hybridoma 1.12.1) was depositedunder terms in accordance with the Budapest Treaty with the AmericanType Culture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209 on Jun. 21, 2005. The hybridoma 1.12.1 has been assigned thefollowing accession number: PTA-6808.

Example 3 Sequences of Anti-ALK-1 Antibodies

To analyze the structure of antibodies produced in accordance with theinvention, nucleic acids were cloned that encode heavy and light chainfragments from hybridomas producing anti-ALK-1 monoclonal antibodies.Cloning and sequencing was accomplished by standard means.

Poly(A)⁺ mRNA was isolated using a Fast-Track™ kit (Invitrogen) fromapproximately 2×10⁵ hybridoma cells for each of the ALK-1 antibodies.cDNA was synthesized from the mRNA by using random primers. The randomprimed cDNA was amplified by PCR using human V_(H) or human Vκ familyspecific variable domain primers in conjunction with primers specificfor the human Cγ2 constant region, or a Cκ constant region to amplifythe antibody variable region including all the framework regions (FRs)and complementarity determining regions (CDRs). Nucleic acid sequenceswere obtained that encode human heavy and kappa light chain transcriptsfrom the anti-ALK-1 producing hybridomas by direct sequencing of bothstrands of PCR products. Sequences were analyzed using Abgenix'sproprietary software and publicly available sequence information forhuman V_(H) and Vκ genes, the “V BASE sequence directory”, Tomlinson etal., MRC Centre for Protein Engineering, Cambridge, UK). Identicalresults could be obtained using publicly available sequence alignmentsoftware by someone of ordinary skill using MacVector and Geneworkssoftware programs.

Specifically, full-length ALK-1 antibody 1.12.1 was cloned intoexpression vectors as follows: Poly(A)⁺ mRNA was isolated using anRNeasy Mini Kit (Qiagen) and cDNA synthesized from the mRNA with theAdvantage RT-for-PCR kit (BD Biosciences) using oligo(dT) priming. Theoligo(dT) primed cDNA for clone 1.12.1 was amplified using primerslisted in Table 2. Amplification was achieved using the Pfu Ultrapolymerase (Stratagene) and a PTC-200 DNA Engine (MJ Research) withcycling as follows: 3′@95° C.; 25× (20″@95° C., 30″@52° C., 1′20″@72°C.); 10′@72° C. Clones were sequence verified using Grills 16^(th)BDTv3.1/dGTP chemistry (Applied Biosystems Inc) and a 3730xl DNAAnalyzer (Applied Biosystems Inc). In the process of cloning 1.12.1V_(H), a silent mutation was introduced in the 8^(th) codon, converting“GGC” to a “GGT.” All sequences were analysed by alignments to the ‘VBASE sequence directory’ (Tomlinson, et al, J. Mol. Biol., 227, 776-798(1992); Hum. Mol. Genet., 3, 853-860 (1994); EMBO J., 14, 4628-4638(1995).)

TABLE 2 Heavy and Light Chain Amplification Primers Used for Cloningfull-length 1.12.1 SEQ Primer ID Name Primer Sequence NO 4-61 5′tcttcaagcttgatatctctagaagccgccaccATGAA 105 ACACCTGTGGTTCTTCCTCC 3′ G1/2_(—) 5′ ttctctgatcagaattcctaCTATTTACCCGGAGACAG 106 FL_R GGAGAGGC 3′A11 5′ tcttcaagcttcccgggagccgccaccATGGAAACCCC 107 AGCGCAGCTT 3′ K_FL_R5′ ttctttgatcagaattctcaCTAACACTCTCCCCTGTT 108 GAAGCTCTTTG 3′Non-hybridizing bases in lower case

Example 4 Gene Utilization Analysis and CDR Analysis

From the nucleic acid sequence and predicted amino acid sequence of theantibodies, the gene usage was identified for each antibody chain. Table3 sets forth the gene utilization of selected hybridoma clones ofantibodies in accordance with the invention.

TABLE 3 Heavy and Light Chain Gene Utilization Heavy Kappa Light ChainGermline Chain Germline SEQ ID SEQ ID Clone NO: V_(H) D_(H) J_(H) NO:V_(K) J_(K) 1.11.1 9 3-33 6-19 JH3B 11 L1 JK4 1.12.1 103 4-31 6-19 JH4B126 A27 JK5 1.13.1 13 4-61 6-19 JH4B 15 A27 JK5 1.14.1 17 4-61 6-19 JH4B19 A27 JK5 1.151.1 21 4-31 3-3 JH3B 23 B3 JK1 1.162.1 25 4-31 JH3B 27A27 JK5 1.183.1 29 4-59 6-19 JH4B 31 L2 JK3 1.31.1 4-31 6-19 JH4B A27JK5 1.8.1 33 4-31 3-3 JH3B 35 B3 JK1 1.9.1 37 3-11 3-22 JH6B 39 A2 JK14.10.1 41 3-15 3-22 JH4B 43 A3 JK4 4.24.1 45 4-31 5-12 JH6B 47 A27 JK54.38.1 49 4-31 4-23 JH4B 51 B3 JK1 4.58.1 53 4-31 4-23 JH4B 55 A27 JK54.62.1 57 4-31 5-12 JH6B 59 A27 JK5 4.68.1 61 4-31 2-2 JH5B 63 A27 JK54.72.1 65 4-31 5-12 JH6B 67 A27 JK5 5.13.1 69 4-31 JH3B 71 A27 JK45.34.1 73 4-31 JH6B 75 A1 JK1 5.53.1 77 3-15 1-1 JH4B 79 B2 JK4 5.56.181 3-11 6-19 JH6B 83 A2 JK1 5.57.1 85 3-11 3-10 JH6B 87 A2 JK1 5.59.1 893-11 6-6 JH6B 91 A2 JK1

Mutagenesis, in the V_(H) (M29I) and V_(κ) (D19A) regions of clone1.12.1, was conducted with the primers listed in Table 4 and theQuickChange kit (Stratagene) according to the manufacturer'sinstructions. The mutated variants were sequence verified and clonedinto expression vectors by standard procedures.

TABLE 4 Mutagenic Oligonucleotides (sequences 5′ to 3′): Primer SenseAntisense 1.12.1 CTCCAGGGGAAAGAG C CACCC CCTACAGGAGAGGGTG G CTCTT (D19A)TCTCCTGTAGG TCCCCTGGAG (SEQ ID NO: 109) (SEQ ID NO: 110) 1.12.1GGTGGCTCCAT C AGCAGTGGT GTAGTATTCACCACTGCT G ATG (M29I) GAATACTACGAGCCACC (SEQ ID NO: 111) (SEQ ID NO: 112) Mutations are indicated inbold and underlined.

Nucleic acid molecules encoding the variable domain of heavy chain (SEQID NO: 5) and the variable domain light chain (SEQ ID: 7) chain of the1.12.1(M29I/D19A) antibody were deposited under terms in accordance withthe Budapest Treaty with the American Type Culture Collection (ATCC),10801 University Blvd., Manassas, Va. 20110-2209 on Jul. 14, 2005. Thedeposits have been assigned the following accession numbers: ATCC No.PTA-6864 for E. coli DH5a containing plasmid pCR2.1 TOPO 1.12.1 V_(H)(M29I):UC 25502; and ATCC No. PTA-6865 for E. coli DH5a containingplasmid pCR2.1 TOPO 1.12.1 V_(κ) (D19A):UC 25503.

A number of anti-ALK-1 specific human antibodies exhibited a commonpattern in CDR1 of the heavy chain variable domain. These lead moleculesutilize the 4-31 or 4-61 heavy chain V-gene segments. The FR1 and CDR1sequences corresponding to these antibody heavy chains are shown inTable 4A, aligned against the germline sequences. A dash (−) in thealignment indicates a residue identical to germline. In all cases, theGYYWS (SEQ ID NO: 136) pattern at the end of CDR1 has undergone somaticmutations to yield a new sequence pattern, whereby the G residue ischanged to an acidic residue (D or E), and the final S residue ischanged to an N in 9 out of the 12 examples. Sequence diversity in otherregions of VH indicates that these are likely to be independent somaticmutation events leading to the same sequence pattern at the end of CDR1of VH.

TABLE 4A ALK-1 Antibody Heavy Chain Sequence Patterns Clone V-geneD-gene J-gene FR1 CDR1 Germline QVQLQESGPGLVKPSQTLSLTCTVS GGSISSGGYYWS(SEQ ID NO: 122) (SEQ ID NO: 123) 5.34.1 VH4-31 -NA- JH6B------------------------- -------D---N 4.58.1 VH4-31 D4-23 JH4B------------------------- -------D---N 4.38.1 VH4-31 D4-23 JH4B------------------------- -------D---- 5.13.1 VH4-31 -NA- JH3B------------------------- -------D---N 1.162.1 VH4-31 -NA- JH3B--------------------I---- -------E---- 4.72.1 VH4-31 D5-12 JH6B------------------------- -------E---- 4.24.1 VH4-31 D5-12 JH6B------------------------- ------ND---N 4.62.1 VH4-31 D5-12 JH6B------------------------- -------D---N 1.31.1 VH4-31 D6-19 JH4B------------------------- -------D---N 1.12.1 VH4-31 D6-19 JH4B------------------------- ---M---E---N GermlineQVQLQESGPGLVKPSETLSLTCTVS GGSVSSGGYYWS (SEQ ID NO: 124) (SEQ ID NO: 125)1.13.1 VH4-61 D6-19 JH4B --H---------------------- -------D---N 1.14.1VH4-61 D6-19 JH4B ------------------------- -------D---N

Example 5 Preparation of 1.12.1 Fab Molecules

Fab fragment of 1.12.1(M29I/D19A) was prepared by digesting1.12.1(M29I/D19A) IgG1 using papain. Protein A purified full length1.12.1(M29I/D19A) IgG1 was incubated with papain (VWR) at 1:50 ratio(papain: protein) in buffer containing 30 mM sodium phosphate (pH 7.0),2 mM EDTA and 2 mM cysteine at 37° C. for 2-3 hours. The digestionmixture was then applied to a protein A mini-column to remove undigestedfull length protein and Fc fragment. Unbound Fab was collected in theflow-through. A size exclusion column (Superdex 200, Amersham PharmaciaBiotech) was then used to further purify the Fab protein and to exchangethe buffer into PBS. Endotoxin was removed by applying the proteinsolution through Detoxi gel (PIERCE) and VIVAPURE® Mini Q ion exchangecolumn (VivaScience) subsequently. The protein was filtered with 0.2 μmsyringe filter and endotoxin level was tested with a LAL pyrogent kit(Cambrex). The final purified protein was at concentration of 2-3 mg/mL,with endotoxin level of <0.1 EU/mg and purity of >95%. 1.12.1(M29I/D19A)Fab fragment has a molecular weight of 47,347 under non-reducedcondition as shown by electron-spray mass spectrometry. Edman N-terminalsequencing analysis confirmed the light chain N-termini sequence ofEIVLTQSPG (SEQ ID NO: 113) and heavy chain sequence of QVQLQESG (SEQ IDNO: 114), respectively.

Example 6 Determination of Avidity Values of Fully Human Anti-ALK-1Monoclonal Antibodies by Surface Plasmon Resonance (SPR) Using BIACORE®

Avidity measures of purified anti-ALK-1 antibodies by surface plasmonresonance using the BIACORE® 3000 instrument were performed as followsusing the manufacturer's protocols.

To perform kinetic analyses, recombinant human ALK-1/Fc fusion protein(hALK-1/Fc) and cynomologus ALK-1/Fc fusion protein (cALK-1/Fc) wereimmobilized on separate flow cells of a CM5 BIAcore sensor chip usingroutine amine coupling. Surfaces were prepared using 10 mM acetatebuffer, pH 5.0 as the immobilization buffer and protein densities of 300and 150 RU were achieved for the hALK-1/Fc and cALK-1/Fc fusionproteins, respectively. Deactivation of unreacted N-hydroxysuccinimideesters was performed using 1 M ethanolamine hydrochloride, pH 8.5.Antibody samples in running buffer were prepared at concentrationsranging from 0.125 to 2 nM (a 0 nM solution comprising running bufferalone was included as a zero reference). Samples were randomized andinjected in duplicate for 10 minutes each across all 4 flow cells usingHBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% SurfactantP20) as running buffer. On-rates were observed to be independent of flowrates from 1 to 100 μL/min indicating no mass transport limitation. Aflow rate of 25 μL/min was used to determine avidity values. Thedissociation of the antibody was monitored for 10 minutes, the surfaceregenerated by a 12 second injection of 100 mM H₃PO₄ (25 μL/min). Theraw data were processed using the Scrubber (©BioLogic Software) softwarepackage and analyzed using the CLAMP (©BioLogic Software) softwarepackage. Multiple data sets from a single surface, six data sets at atime, were simultaneously fit globally to a simple 1:1 Langmuir bindingmodel utilizing a common variable Rmax value. Table 5 lists avidityvalues for representative anti-ALK-1 antibodies of the presentinvention. The represented data indicate that the antibodies prepared inaccordance with the invention possess high affinities and strong bindingconstants for human ALK-1.

TABLE 5 Determination of Avidity Value by Surface Plasmon Resonance(BIACORE ®) hALK-1/Fc hALK-1/Fc cALK-1/Fc Clone Avidity(pM) k_(off)(1/s) Avidity (pM) 1.12.1(M29I/D19A) <6.8 <5.0 × 10⁻⁶   27 1.14.2 76 5.6× 10⁻⁵ 280 1.27.3 2.9 1.9 × 10⁻⁵ 60 1.31.1 <13 <5.0 × 10⁻⁶   150 1.162.118 1.1 × 10⁻⁵ 62 1.183.2 220 3.1 × 10⁻⁵ 1800 4.24.2 70 4.4 × 10⁻⁵ 4304.38.1 100 4.0 × 10⁻⁵ 150 4.58.2 40 1.6 × 10⁻⁵ 130 4.62.1 9.6 7.6 × 10⁻⁶19 4.68.2 86 3.8 × 10⁻⁵ 320 4.72.2 73 3.4 × 10⁻⁵ 280 5.13.3 91 6.3 ×10⁻⁵ 190 1.12.1(M29I/D19A) refers to the mAb 1.12.1 variant that wasexpressed recombinant mAb containing two specific amino acid mutations(methionine at position 29 in the heavy chain replaced with isoleucineand aspartic acid at position 19 in the light chain replaced withalanine).

Determination of Avidity Value by Surface Plasmon Resonance(BIACORE®)Example 7 Determination of Affinity Constants (K_(D)) of Variants ofFully Human Anti-ALK-Monoclonal Antibody 1.12.1 by Surface PlasmonResonance (SPR) using BIACORE®

Affinity measures of purified anti-ALK-1 antibodies by surface plasmonresonance using the BIACORE® 3000 instrument were performed as followsusing the manufacturer's protocols.

To perform kinetic analyses, variants of fully human anti-ALK-1monoclonal antibody 1.12.1 were immobilized onto the dextran layer of aCM5 biosensor chip using amine coupling. Surfaces were prepared using 10mM acetate buffer pH 5.0 as the immobilization buffer and proteindensities of 3500-4800 RU were achieved. Deactivation of unreactedN-hydroxysuccinimide esters was performed using 1 M ethanolaminehydrochloride, pH 8.5. Samples of monomeric ALK-ECD in running bufferwere prepared at concentrations ranging from 2.63 to 640 nM (a 0 nMsolution comprising running buffer alone was included as a zeroreference). Samples were randomized and injected for 2 minutes eachacross all 4 flow cells using HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3mM EDTA, 0.005% Surfactant P20) as running buffer. A flow rate of 25μL/min was used to determine affinity constants. Dissociation ofmonomeric ALK-ECD was monitored for 10 minutes, the surface regeneratedby a 12 second injection of 100 mM H₃PO₄ (25 μL/min). The raw data wereprocessed using the Scrubber (©BioLogic Software) software package andanalyzed using the CLAMP (©BioLogic Software) software package. The datawere fit globally to a simple 1:1 Langmuir binding model. Table 6 listsaffinity measurements for variants of human anti-ALK-1 monoclonalantibody 1.12.1 of the present invention.

TABLE 6 Determination of mAb 1.12.1 variant affinity constant, K_(D), bysurface plasmon resonance (BIACORE ®) Antibody on-rate (M⁻¹ s⁻¹)Off-rate (s⁻¹) K_(D) (nM) 1.12.1 1.9 × 10³ 7.4 × 10⁻⁵ 39 1.12.1(rWT) 2.2× 10³ 5.8 × 10⁻⁵ 26 1.12.1(D19A) 2.6 × 10³ 4.4 × 10⁻⁵ 17 1.12.1(M29I)2.4 × 10³ 9.1 × 10⁻⁵ 38 1.12.1(M29I/D19A) (1)* 2.2 × 10³ 9.5 × 10⁻⁵ 431.12.1(M29I/D19A) (2)* 2.3 × 10³ 8.4 × 10⁻⁵ 37 *The two affinityconstants for 1.12.1(M29I/D19A) (1) and (2) were obtained using twoseparate surfaces. 1.12.1 refers to the mAb 1.12.1 variant that wasisolated from the hybridoma. 1.12.1(rWT) refers to the mAb 1.12.1variant that was expressed recombinant mAb. 1.12.1(M29I) refers to themAb 1.12.1 variant that was expressed recombinant mAb containing aspecific single amino acid mutation where the methionine at position 29in the heavy chain was replaced with isoleucine. 1.12.1(D19A) refers tothe mAb 1.12.1 variant that was expressed recombinant mAb containing aspecific single amino acid mutation where the aspartic acid at position19 in the light chain was replaced with alanine. 1.12.1(M29I/D19A)refers to the mAb 1.12.1 variant that was expressed recombinant mAbcontaining two specific amino acid mutations (methionine at position 29in the heavy chain replaced with isoleucine and aspartic acid atposition 19 in the light chain replaced with alanine).

Determination of mAb1.12.1 variant affinity constant, K_(D), by surfaceplasmon resonance (BIACORE®) Example 8 Determination of AffinityConstants (K_(D)) of Representative Fully Human Anti-ALK-1 MonoclonalAntibodies by Surface Plasmon Resonance (SPR) using BIACORE®

Affinity measures (K_(D) and k_(off)) of purified anti-ALK-1 antibodiesby surface plasmon resonance using the BIACORE® 3000 instrument wereperformed as follows using the manufacturer's protocols.

To perform kinetic analyses, affinity-purified mAbs were immobilizedonto the dextran layer of a CM5 biosensor chip using amine coupling.Surfaces were prepared using 10 mM acetate buffer pH 5.0 as theimmobilization buffer and protein densities of 200-400 RU were achieved.Deactivation of unreacted N-hydroxysuccinimide esters was performedusing 1 M ethanolamine hydrochloride, pH 8.5. Samples of monomericALK-ECD in running buffer were prepared at concentrations ranging from3.125-400 nM (a 0 nM solution comprising running buffer alone wasincluded as a zero reference). Samples were randomized and injected induplicate for 2 minutes each across all 4 flow cells using HBS-EP (10 mMHEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20) as runningbuffer. On-rates were observed to be independent of flow rates from 1 to100 μL/min indicating no mass transport limitation. A flow rate of 25μL/min was used to determine affinity constants. Dissociation ofmonomeric ALK-ECD was monitored for 10 minutes, the surface regeneratedby a 12 second injection of 100 mM H₃PO₄ (25 μL/min). The raw data wereprocessed using the Scrubber (©BioLogic Software) software package andanalyzed using the CLAMP (©BioLogic Software) software package. The datawere fit globally to a simple 1:1 Langmuir binding model. Table 7 listsaffinity measurements for representative anti-ALK-1 antibodies of thepresent invention:

TABLE 7 Determination of Affinity Constant, K_(D), for RepresentativeMonoclonal Antibodies by Surface Plasmon Resonance (BIAcore) mAb on-rate(M⁻¹ s⁻¹) off-rate (s⁻¹) K_(D) (nM) 1.12.1(M29I/D19A) 3.3 × 10⁴ 8.2 ×10⁻⁴ 25 1.31.1 3.2 × 10⁴ 1.9 × 10⁻⁴ 6.0 4.72.1 3.2 × 10⁴ 2.5 × 10⁻⁵ 0.8Fab 1.12.1(M29I/D19A) 3.8 × 10⁴ 8.2 × 10⁻⁴ 22 The monomeric ALK-ECD usedto generate the data in Example 8 was a different preparation than thatused to generate the data in Example 7. 1.12.1(M29I/D19A) refers to themAb 1.12.1 variant that was expressed recombinant mAb containing twospecific amino acid mutations mentioned above (aspartic acid at position19 in the light chain replaced with alanine and methionine at position29 in the heavy chain replaced with isoleucine). Fab 1.12.1(M29I/D19A)refers to the Fab fragment of mAb 1.12.1(M29I/D19A) prepared bydigesting 1.12.1(M29I/D19A) IgG1 using papain.

Determination of Affinity Constant, (K_(D)) for RepresentativeMonoclonal Antibodies by Surface Plasmon Resonance BIACORE® Example 9Identification of Epitope Selectivity of Anti-ALK-1 Antibodies

Cross-competition experiments were performed using the BIACORE® 3000instrument (Biacore International AB, Uppsala, Sweden and Piscataway,N.J.), following the manufacturer's protocols.

Recombinant human ALK-1/FC chimera was immobilized onto the dextranlayer of a CM5 biosensor chip using amine coupling. Chips were preparedusing 10 mM acetate buffer pH 5.0 as the immobilization buffer and aprotein density of 940 RU was achieved. Deactivation of unreactedN-hydroxysuccinimide esters was performed using 1 M ethanolaminehydrochloride, pH 8.5.

Purified mAbs were diluted to a concentration of 50 nM in HBS-EP runningbuffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Polysorbate20). A primary antibody was chosen and then injected across the flowcell for 600 seconds at a rate of 10 μL/min. After the injection wascomplete, a secondary antibody was chosen and injected across the sameflow cell for 600 seconds at a rate of 10 μL/min. The sensor surface wasregenerated by a 12 second injection of 100 mM H₃PO₄ (25 μL/min).

After regeneration, the primary antibody was again injected across theflow cell for 600 seconds at a rate of 10 μL/min. After the injectionwas complete, a different secondary antibody was chosen and injectedacross the same flow cell for 600 seconds at a rate of 10 μL/min. Oncethe entire panel of 14 antibodies had been used as the secondaryantibody a new primary antibody was chosen and the procedure repeatedwith the new primary antibody. These procedures were carried out untilall possible combinations of primary and secondary antibodies had beeninjected across the flow cell. Binding of the secondary antibody wasconsidered to have occurred if the total response observed afterinjecting both antibodies exceeded that observed for both possiblethreshold values. Threshold values were determined by using the sameantibody as both the primary antibody and secondary antibody. Shown inTable 8, a response matrix was created based on whether binding wasobserved: - indicates no binding of the secondary antibody, x indicatesbinding was observed (response was greater than the threshold values forthe individual antibodies). Grouping the clones that show the samereactivity pattern gives rise to two different epitope bins with 1.11.1in one bin and all the other antibodies in the other bin.

TABLE 8 BIACORE ® epitope Dinning response matrix

BIACORE® epitope binning response matrix Example 10 Isolation ofCynomolgous Monkey ALK-1 Gene

Cynomolgus monkey (“Cyno”) ALK-1 gene was extracted from Cyno lungtissue. Based on the published gene sequence for human ALK-1 (Genebankrecord L17075), primers were designed to PCR amplify the full-lengthCynomolgus ALK-1. mRNA was prepared from frozen excised cynomolgus lungtissue (ca. 1 g) using the mRNA purification kit (Ambion, Catalog No.1915) according to the manufacturer's instructions. 200 ng of the mRNAwas reverse transcribed and PCR amplified using the OneStep RT-PCR kit(Qiagen, Catalog No. 210210) utilizing gene-specific oligos:5′-AGCGGGCCCAGAGGGACCATG (Seq ID NO: 115) (forward) and5′-CAGAAAGGMTCAGGTGCTCCTGGGCTA (Seq ID NO: 116) (reverse) at anannealing temperature of 61° C. An RT-PCR product of the appropriatesize (˜1.5 Kb) was excised and purified from a 0.9% agarose gel afterelectrophoresis, then TOPO-TA cloned into the pCR4-TOPO vector(Invitrogen, Catalog No. K4575-01). The insert was sequenced to obtainthe ORF nucleotide sequence of Cynomolgus ALK-1. The nucleotide andpredicted translated amino acid sequences are shown in SEQ ID NOs: 93and 94, respectively. While the cytoplasmic portion of the gene encodesidentical protein sequences between Cyno and human, there are 5 aminoacid differences in the extracellular domain (ECD, which includespositions 22-118) and 1 amino acid difference in the transmembranedomain of the protein. ECD sequence identity between human and Cyno is94.8%. An alignment of the human and primate ECD is shown in FIG. 2.

A pair of primers (forward: 5′-GATTATGGCCTTGGGCTCCCCCAGGAAA (Seq ID NO:117) and reverse: 5′-GGGCTATTGAATCACTTTAGGCTTCTCTGGACTGTTG) (Seq ID NO:118) were used to PCR amplify the full-length Cynomolgus ALK-1 gene.

Example 11 Determination of Cell Surface Binding Characteristics andPrimate Crosshybridisation by Flow Cytometry (FACS)

To generate ALK-1-overexpressing cell lines, which can be used to testanti-ALK-1 binding affinity using flow cytometry (FACS), full-lengthhuman, Cyno, and rat ALK-1 genes were cloned into Invitrogen's (CatalogNo. K6510-20) pcDNA5/FRT/To TOPO vector and transfected into 293 Flp-InT-Rex host cell (Invitrogen, Catalog No. R780-07), respectively.Selections were carried out using hygromycin to obtain the final stablecell lines. Overexpressing of the respective full-length ALK-1 proteinswere achieved by tetracycline (2 μg/mL) induction at 37° C./5% CO₂ for24 hours.

Anti-ALK-1 mAbs were tested for their binding affinities to cell surfaceALK-1 using FACS assay, employing 293 stable cells overexpressing ALK-1proteins. The cells were detached using trypsin-EDTA and washed withcold PBS-SA. After being aliquoted into 96-well plates, the cells wereblocked by serum and incubated with different concentrations of specificmAb for 1 hour at 4° C. Subsequently, the cells were washed andincubated with an anti-human κ secondary antibody conjugated with theR-PE fluorophore before analyzed using a FACSCalibur flow cytometer (BDBiosciences). 10,000 events were collected for each sample withoutapplying any gating. Shown in Table 9, the geometric mean of each samplehistogram was plotted as a function of the mAb concentration and K_(D)was calculated for each mAb after fitting to a two-state equilibriummodel. Examples of equivalent human and primate FACS experiments areshown in FIG. 3.

TABLE 9 Mean Binding Affinity (K_(D)) Results of Anti-ALK-1 MonoclonalAntibodies to Cell Surface Human or Cyno ALK-1 Measured by FACS K_(D)(nM) Antibody Human Cyno 1.12.1 6.7 2.0 1.27.1 3.7 2.2 1.162.1 5.6 3.04.38.1 9.3 3.4 4.58.1 14.0 6.7 4.72.1 6.4 3.8 5.13.1 3.2 1.6 1.31.1 3.21.7 4.24.1 7.6 3.1 4.62.1 2.3 0.78 4.68.1 8.4 9.0 In addition, 1.12.1was shown by the FACS assay to have very limited cross-over to rat(K_(D) > 100 nM) and is predicted to have very low cross-over to mousein view of 74% and 68% ECD sequence identity between rat/human andmouse/human ALK-1, respectively).

FACS assay was also used to determine K_(D) of the recombinant 1.12.1mAb variants. Shown in Table 10, the results indicate similar bindingaffinity from the recombinant antibody.

TABLE 10 Mean Binding Affinity (K_(D)) Results of 1.12.1(M29I/D19A)Variants to Cell Surface Human or Cyno ALK-1 Measured by FACS K_(D) (nM)Antibody Human Cyno 1.12.1 6.7 2.0 1.12.1(rWT) 5.9 6.0 1.12.1(M29I) 6.03.3 1.12.1(D19A) 5.7 3.8 1.12.1(M29I/D19A) 7.2 3.4 Fab 1.12.1(M29I/D19A)0.77 ND 1.12.1 refers to the mAb 1.12.1 variant that was isolated fromthe hybridoma. 1.12.1(rWT) refers to the mAb 1.12.1 variant that wasexpressed recombinant mAb. 1.12.1(M29I) refers to the mAb 1.12.1 variantthat was expressed recombinant mAb containing a specific single aminoacid mutation where the methionine at position 29 in the heavy chain wasreplaced with isoleucine. 1.12.1(D19A) refers to the mAb 1.12.1 variantthat was expressed recombinant mAb containing a specific single aminoacid mutation where the aspartic acid at position 19 in the light chainwas replaced with alanine. 1.12.1(M29I/D19A) refers to the mAb 1.12.1variant that was expressed recombinant mAb containing two specific aminoacid mutations (methionine at position 29 in the heavy chain replacedwith isoleucine and aspartic acid at position 19 in the light chainreplaced with alanine). Fab 1.12.1(M29I/D19A) refers to the Fab fragmentof mAb 1.12.1(M29I/D19A) prepared by digesting 1.12.1(M29I/D19A) IgG1using papain.

Example 12 TAQMAN® Assay for Id1

HUVECs (Biowhittaker, Cat. # CC-2519) were seeded on 24-well plates,12000 cells/well in 600 μL of complete HUVEC medium (EGM-2 Bullet kit,Biowhittaker, Cat. # CC-3162), and allowed to grow overnight. Thefollowing day the cells were typically 50% confluent. Complete Mediumwas removed and200 μL of Starvation Medium (EBM-2 with 0.2% FBS only)was added. Cells were incubated for 2 hrs. Then the cells were treatedwith 40 μL of antibody solution in PBS. Lyophilized Ab was reconstitutedwith sterile PBS. Finally, the cells were treated with 1% FBS/Basalmedium (final concentrations) for 30 minutes, the medium was removed andthe cells were lysed in 400 μL of RTL Buffer (Rneasy 96 kit, Qiagen,Cat. # 74182), according to the manufacturer's protocols. Subsequently,RNA was prepared using RNeasy kit (according to manufacturer'sinstructions). The RNA was eluted and quantitated with RIBOGREEN® RNAQuantitation Kit (Molecular probes, Cat. # R-11490). Equal amount oftotal RNA was used for real time PCR analysis to detect Id1 RNAexpression (ABI 7900 instrument). PCR was conducted using the TAQMAN®One Step PCR Master Mix Kit (ABI, Cat. # 4309169) and the ID1primer/Probe sequences listed below. PCR was conducted using 40 cyclesof the following annealing and amplification conditions: 95° C., 15seconds; 60° C., 1 min.

TAQMAN ® probe: CPG-conjugated 5′-6-FAM, and 3′-TAMRA. Name: ID1-ProbeSequence: 5′ CCAGCACGTCATCGACTACATCAGGGA 3′ (Seq ID NO: 119) TAQMAN ®PCR Primers: Name: ID1-F Sequence: 5′ AAGGTGAGCAAGGTGGAGATTC 3′ (Seq IDNO: 120) Name: ID1-R Sequence: 5′ TTCCGAGTTCAGCTCCAACTG 3′ (Seq ID NO:121)

Examples of Id1 titrations for the 1.12.1(M29I/D19A) lead molecule(including 1.12.1 sequence variants) and the Fab derivative are shown inFIGS. 4 and 5.

A summary of mean IC₅₀ values for this assay is shown in Table 11. AllIC₅₀ determinations were run in triplicate.

Example 13 Smad1 Phosphorylation Detected by ODYSSEY® Infrared ImagingSystem from L1-COR Biosciences (24-Well Plate)

HUVECs (Biowhittaker, Cat. # CC-2519) were seeded on 24-well plates,18000 cells/well in 600 μL of complete HUVEC medium (EGM-2 Bullet kit,Biowhittaker, Cat. # CC-3162), and allowed to grow overnight. Thefollowing day the cells were typically 50% confluent. Complete Mediumwas removed and200 μL of Starvation Medium was added (Starvation Medium:EBM-2 with 0.2% FBS only). Cells were Incubated for 2 hrs. Then thecells were treated with 40 μL of antibody solution in PBS for 3 hours.Finally, the cells were treated with 0.3× Complete Medium (finalconcentration) for 35 minutes. The medium was removed, and the cellswere lysed in 80 μL of 1.1× Sample Buffer (Invitrogen, Cat. No.NP0007).Phosphorylated Smad1 was determined by Western Blotting using XCELLSURELOCK® Mini-Cell & Blot Module (Invitrogen, Cat. # EI0002).Phosphorylated Smad1 was detected using rabbit anti-phosphor-Smad1antibody (Cell Signaling, Cat. No. 9511), which is then detected byIRDYE® 800 Conjugated Anti-RABBIT IgG (Rockland Immunochemicals, Cat.No. 611-732-127). Amount of phosphorylated Smad1 was quantified usingODYSSEY® Infrared Imager (Li-Cor). Actin (Santa Cruz, # sc-8432) wasused for normalization (anti-mouse Alex 680, Molecular Probe, Cat. No.A-21058). A summary of mean IC₅₀ values for this assay is shown in Table11. All IC₅₀ determinations were run in triplicate.

TABLE 11 ID1 TAQ- pSmad1 MAN ® Western Clone IC₅₀ nM IC₅₀ nM 1.11.1 ndnd 1.12.1(M29I/D19A) 16 18 1.13.1 100 87 1.27.1 82 70 1.29.1 94 821.31.1 24 21 1.162.1 75 15 1.183.1 58 17 4.24.1 100 82 4.38.1 87 524.58.1 14 15 4.62.1 24 34 4.68.1 141 110 4.72.1 21 35 5.13.1 30 68

Example 14 Internalization Characteristics of Anti-ALK-1 MonoclonalAntibodies

FACS was used to monitor the time course of the remaining cell-surfacereceptor ALK-1 as well as the neutralizing antibody. Remainingcell-surface ALK-1 is monitored by a marker antibody which is capable ofbinding cell surface ALK-1 yet recognizing a different epitope from theneutralizing antibody. A mouse anti-human ALK-1 ECD mAb (R&D systems,Cat. No # AF310) was identified and used in the study as the markerantibody.

Time course of internalization was studied using endothelial cell linesHUVEC and HUAEC. The cells were grown at 37° C. with 5% CO₂ in 24-wellplates containing 200 μL of complete culture medium per well. At each of11 time points over the course of 48 hours, 2 μL of 1 mg/mL antibodysolution was added to one well and mixed (final concentration of theneutralizing antibody is 10 μg/mL). The plate was then put back into the37° C. incubator until the 0 hour time point, when the plate was placedon ice to stop the internalization process. Marker antibody was added tothe wells at this point (10 μg/mL final concentration) and incubated onice for 1 hr. The cells were then washed with PBS detached bytrypsination and transferred into a 96-well plate. Cells were thenwashed, blocked, and treated with secondary antibodies bearing differentflurophores in order to monitor both neutralizing antibody and receptorALK-1 remaining on the cell surface. The samples were assayed on aFACSCalibur flow cytometry instrument, counting 3,000-5,000events/sample. The Geometric Mean of each sample in the specificfluorescence channel was calculated and plotted as a function of time.The data were fitted to a modified radio-decay equation to obtain thehalf-time (t_(1/2)) of internalization as well as the percentage ofneutralizing antibody or receptor ALK-1 remaining on the cell surfacewhen the internalization reached steady-state. As shown in FIG. 6, mAb1.12.1(M29I/D19A) internalizes at the same rate and to the same extendas the cell surface receptor ALK-1. Half-life of the 1.12.1(M 29I/D 19A)internalization is ˜2 hr. An equilibrium was reached when 50% of theantibody was internalized. A polyclonal antibody purchased from R&Dsystems (Cat. No # AF370) internalizes at a t_(1/2) of 1 hr and reachesthe steady-state with ˜70% of the receptor being internalized (FIG. 6).Similar internalization characteristics were observed with other humananti-ALK-1 mAbs of the invention (not shown).

Example 15 Establishment of Human Foreskin—SCID Chimera Mice

Significant modification of the surgery procedure was made to aprocedure published previously by H-C Yan, et al “Human/Severe CombinedImmunodeficient Mouse Chimeras, An Experimental In Vivo Model System toStudy the Regulation of Human Endothelial Cell-Leukocyte AdhesionMolecules”, J. Clin. Invest. 91:986, 1993; J. Varner “Regulation ofAngiogenesis in Vivo by Ligation of Integrin a5b1 with the CentralCell-Binding Domain of Fibronectin” Amer. J. Path. 156 (4):1345, 2000;K. Tahtis, et al “Expression and Targeting of Human FibroblastActivation Protein in a Human Skin/Severe Combined Immunodeficient MouseBreast Cancer Xenograft Model” Mol. Cancer. Ther. 2(8):729, 2003. Uponarrival from National Disease Research Institute and Cooperative HumanTissue Network, human foreskin pieces were trimmed of unhealthy regionsand transferred to RPMI media (CELLGRO®/Mediatech, Cat# MT-15-040-CVsupplemented with Penicillin and Streptamycin (GIBCO®/Life Tech, Cat#15070-063) (add 5 mLs of the pen/strep stock solution into 500 mLs ofRPMI). Using a scalpel and cutting in sterile petri dish, the skins weretrimmed to an oval shape of approximately 8×13 mm cleaning any raggedends and connective tissues, and stored on wet ice prior to surgery. Theappropriate volume (4 μL/gram of animal) of 100 mg/mL(KeTASET®(ketamine), Fort Dodge Animal Health)/1 mg/mL medetomidine(Pfizer Animal Health-DORMITOR®(medetomidine)) solution wasintraperitoneally injected into scid mouse abdomen (i.e., at a 45°angle, under skin but not too deep internally). Once anesthetized, themice were applied with eye lubricant, a subcutaneuosly injection ofKetoprofen (10 mg/kg, Fort Dodge Animal Health) and hair was shaved overthe site of the surgery. The surgical region was surgically scrubbedthree times using the Chlorhexiderm (Butler, Chclo-Scrub 40, cat #WAB20109) and then the alcohol in a circular motion that started fromthe center of the surgical site outward and avoided going from a dirtyarea back into a clean area. The mice were transferred to the preparedsurgical hood and placed on the heated water pad (Gaymar Industries,cat# TP500 T/Pump) that was maintained at 37° C. The mice were thenplaced under isofluorin anesthesia for the duration of the surgery. Thedorsal side of a mouse was covered with a surgical drape cut to exposethe surgery site. The mouse skin was picked up with forceps and an ovalshaped skin tissue was cut with curved scissors with one motion. Anappropriate sized human foreskin was placed on the mouse. The human andmouse skin were sutured together using the ETHILON® suture (Ethicon cat#697H.), starting at top of oval, then the bottom, then the farthestright and then farthest left side. More stitches were made in between tofurther secure the tissues together. Approximately 8 stitches were madeequal-distantly around the skin. During the surgery, used a syringe withsterile saline to irrigate the skin/mouse surgical wound when it becamedry. A BANDAID® was placed over the wound. A transparent dressing (3MTEGADERM®) was then used to loosely wrap around the bandage. Thedressing was cut to size to cover an area slightly wider than theBANDAID® . The mouse was then given Atipamezole (50-100 μL, PfizerAnimal Health-ANTISEDAN®(atipamezole)) and the mouse recovered in aheated cage in 5-10 min. The dressing and the bandages were removed in7-10 days and by 15^(th) day most of the skins appeared as scabs.Complete healing occurred between 21-28 days after which time skins wereready to be inoculated with tumor cells. Shown in FIG. 8 is an exampleof the histological (H & E Staining) analysis of a section of theengrafted skin post surgery. The histology of the engrafted skin closelymimics the characteristics of human skin implanted in mice described byTahtis, et al “Expression and Targeting of Human Fibroblast ActivationProtein in a Human Skin/Severe Combined Immunodeficient Mouse BreastCancer Xenograft Model” Mol. Cancer. Ther. 2(8):729, 2003. h.e.: humanepidermal layer; h.d.: human dermal layer.

Example 16 Collagen Model in Human Foreskin—SCID Chimera Mice

Collagen I stock solution (cat# 354236, Becten-Dickinson) was diluted to4 mg/mL with 0.02 N acetic acid and was kept on ice before implantation.The acidic collagen solution (8 parts) was mixed with 10×M199 (Sigma,Cat# M9163) (1 part) and human plasma fibronectin (Fn) (cat# 354008,Becten-Dickinson) to reach a final Fn concentration of 90 μL/mL; NaOH(1.0 N) was added to adjust pH to ˜7.2. The Collagen/Fn mixture was kepton ice until use. The implant mix was prepared using the aboveCollagen/Fn mixture plus angiogenic inhibitor of interest with orwithout human macrovescular endothelial cells (HMVEC), (CascadeBiologics, Cat# C-010-5C). The HMVECs were prepared as 6×10⁶ cells/mL inPBS. 50-100 μL of the implant mixture was injected intradermally intothe foreskin in the scid chimera mouse. 7-14 days later, the collagenplugs were harvested, embedded in the OCT compound (cat# 4583, SajuraFinetek, CA) and snap frozen for immunohistochemistry analysis. Thecollagen plug in the foreskin was identified with the Trichrome Kit(cat# KCl 641, Mater Tech, CA) as blue staining as shown in FIG. 9 (A).Human vessels were identified by staining for human P-CAM using theanti-human CD-31 antibody (Clone 13.3, Vector Laboratories) (FIG. 9(B)). Table 12 summarizes the human vessel staining and quantificationin the collagen model in the foreskin—SCID chimera mice.

TABLE 12 Summary of the Collagen model results % of Human vessel ControlHMVEC Treatment Days of Study End scoring (human Matrix in Matrix (Rx)Rx Point (1 × 10³) vessels) 1.6 mg/ml None no treatment 4 human CD-310.036 ± 0.001 40 Collagen Staining 1.6 mg/ml   7 × 10³ no treatment 40.071 ± 0.022 78 Collagen 1.6 mg/ml 1.4 × 10⁴ no treatment 4 0.063 ±0.016 69 Collagen 2.4 mg/ml None no treatment 4 0.091 ± 0.056 100Collagen 2.4 mg/ml   7 × 10⁴ no treatment 4 0.067 ± 0.049 74 Collagen2.4 mg/ml 1.4 × 10⁴ no treatment 4 0.062 ± 0.047 68 Collagen 3.0 mg/ml8.8 × 10³ Non- 4 human CD-31 54 ± 9  100 Collagen treatment Staining 3.0mg/ml 8.8 × 10³ Isotype 4 52 ± 13 96 Collagen control antibody 100 μg/mlmixed in gel 3.0 mg/ml 8.8 × 10³ 1.12.1(M29I/ 4 15 ± 3  28 CollagenD19A) antibody 100 μg/ml mixed in gel 3.0 mg/ml none no treatment 4human CD-31 0.112 + 0.026 100 Collagen Staining 5.0 mg/ml none notreatment 4 0.031 + 0.012 28 Collagen 3.0 mg/ml none Isotype 4 humanCD-31 75 ± 15 100 Collagen control Staining antibody 100 μg/ml, id.Injection 3.0 mg/ml none 1.12.1(M29I/ 4 39 ± 11 52 Collagen D19A)antibody 100 μg/ml, id. injection 3.0 mg/ml none 1.14.1 4 44 ± 28 59Collagen antibody 100 μg/ml, id injection

Example 17 M24met Tumor Model in Human Foreskin —SCID Chimera Mice

Typically graft age of between 5-10 weeks post surgery were used inthese studies. The M24met cell line was described by Mueller andcoworkers in “Tissue factor-initiated thrombin generation activates thesignaling thrombin receptor on malignant melanoma cells”, CancerResearch, 55(8):1629-32, 1995. M24met cell suspension was prepared asfollowing: 80% confluent M24met cells were washed, typsonized usingTrpsin/EDTA (Gibco, Cat# 25200-056) and collected in the PRMI(CELLGRO®/Mediatech, cat# MT-15-040-CV) media supplemented with 10% FBS(CELLGRO®/Mediatech, Cat# AKD-11775) and 2 mM L-glutamine(CELLGRO®/Mediatech, Cat# 25-005-CI). The cells were centrifuged at 600rpm for 5 min,resuspended in sterile PBS. Cell counts were estimatedusing Coulter Counter (Beckman Coulter, Model Z2). The Cells werecentrifuged at 600 rpm for 5 min and were re-suspended in Collagen/andFn (3 mg/ml) mixture to obtain a 4×10⁷ cells/ml cell suspension forimplantation.

To inoculate, 2×10⁶ above cells were injected (50 μl of 4×10⁷ cells/ml)intradermally into the engrafted human skin in the mouse. At day 5-7post implant, the tumors would be palpable and the mice were randomizedinto the Control and the Treatment groups before the dosing would start.The Control group is defined as such that the animals would receiveeither no dose, dose of the Vehicle in which the anti-ALK-1 antibody wasconstituted, or dose of the isotype matched IgG₂ human monoclonalantibody anti-KLH (Pfizer Inc). The Treatment group is defined as suchthat the animals would receive a dose of the anti-ALK-1 antibody 1.12.1(M29I/D19A).

Example 18 Human and Mouse CD-31 Immunofluorescence (IF) Dual Staining

The frozen tissue sections were air-dried and fix at −20° C. in acetone(Fisher, Cat#A16S-4), or 10 min. The samples were air dried again andwashed in PBS three times at 5 min each. The samples were blocked in 5%rabbit serum (Vector Laboratories, Cat# S-5000) in PBS fro 30 min atroom temperature. Primary antibody mixture was prepared in 5% rabbitserum with the anti-human CD-31 antibody (Santa Cruz, Cat# SC1505) andthe anti-mouse CD-31 (Pharmingen, Clone Mec1 3.3, Cat# 01951A) at 1:100and 1:150 dilutions, respectively. The above antibody mixture was addedto the tissue samples for 1 hour at RT. The blocks were washed in PBSfor three times at 5 min each before incubated with the secondaryantibody mixture for 1 hour at RT. The secondary antibody mixture wasprepared in PBS/0.05% Tween-20 (Sigma, Cat# P1379), Texas Red rabbitanti-goat antibody (Jackson Labs, Cat# 305-075-003) and FITC rabbitanti-Rat antibody (Jackson Labs, Cat# 312-095-003). The antibodies werediluted at 1:50 if frozen antibodies were used or at 1:100 if freshantibodies were used. The slides were washed again in PBS for threetimes at 5 min each before mounted in VECTASHIELD® (Hard Set, Mountingmedium with DAPI, Vector Lab, CA, Cat# H-1500). The slides were kept indark and 4° C. until image analysis. The image analysis was performedusing an Olympus BX60 fluorescent microscope and photographs were takenusing an Olympus microfire digital color camera. Pictures from 3-5 hotspots/slide, one slide/animal, 4-7 animals/group were taken and thevessel areas as indicated by positive staining of anti-human CD-31 werequantified by three individuals using Image Pro Plus v4.5(MediaCybernetics). The pharmacodynamic end point (group mean) wasexpressed as either the percent of human CD-31 inhibition compared tothe Control group or as total human vessel area. Statisticalsignificance was determined by ANOVA. Shown in FIG. 10 is animmunofluorescent image of human(red) and mouse (green) vessels of theM24met tumor in the human foreskin SCID chimera mouse.

Example 19 Human CD-31 Immunohistochemistry (IHC) Staining

The frozen tissue sections were air-dried and fix at −20° C. in acetonefor 10 min. The samples were air dried again and washed in PBS twice at5 min each. The samples were incubated in 0.075% H₂O₂/methanol (FisherCat# A433-4) for 15 min and wash in PBS three times at 5 min each. Thesamples were blocked in 5% rabbit serum/PBS for 30 min and applied withanti-human CD-31 antibody 1:100 (Santa Cruz, Cat# SC1505) in 5% rabbitserum for 1 hour at RT. The samples were washed in PBS twice at 5 mineach and applied with rabbit anti-goat at 1:200 (Vector Labs, Cat#BA-5000) in 5% rabbit serum for 35 min at RT. The slides were thenwashed in PBS twice at 5 min each and freshly made streptavidin (VectorLabs, ABC Elite kit, Cat# PK-6100) was added. The slides were washedagain in PBS twice at 5 min each and then developed in diaminobenzidine(DAB) (Vector Labs, Cat#SK-4100). The slides were wash in PBS twice at 5min each followed by Mayers haematoxylin (Sigma, Cat# HHS-32) for 5seconds. The samples were rinsed well in diH₂O and dipped twice brieflyin the diluted (5 ml stock in 1 L of diH₂O) ammonium hydroxide solution(Sigma, Cat# A-6899) and rinsed in diH₂O again. The samples were thendehydrated in the 70%, 90% and then 100% alcohol (Harleco, Cat#65347/85) 1 minute each and finally in xylene (J T Baker, Cat# 516,09).The slides were mounted with Cytoseal 60 (Stephens Scientific, Cat#8310-4,) and covered with cover slips for image analysis. Shown in FIG.11 is the IHC image of human vessels (brown) of the M24met tumor in thehuman foreskin-SCID chimera mouse.

Example 20 Therapeutic Treatment with the Anti-ALK-1 Antibody 1.12.1(M29I/D19A)

For treatment, the dosing was performed either subcutaneously (sc) orintravenously (iv). Typically one dose of the 1.12.1(M29I/D19A) antibodywas given for each study. The second dose of the ALK-1 antibody, ifnecessary, was administered on day 9 or 10. Some times multiple doselevels, i.e., 1, 5, 10, 50 mg/kg, were administered to investigatedose-dependent inhibition of human vessel growth. Animals were monitoreddaily and tumors were measured three times/week by calipers. By day14-17 the tumors were between 250-350 mm³ and were removed from themice, embedded in OCT and frozen down for IF or IHC analysis. Shown inFIG. 12 are representative immunofluorescent images of human (red) andmouse (green) vessels of the Control and 1.12.1(M29I/D19A) Treated (10mg/kg) M24met tumors in the human foreskin scid chimera mouse.Dose-dependent inhibition of human tumor vessels by 1.12.1(M29I/D19A) inthe human foreskin SCID chimera mouse model is shown in FIG. 13 and asummary of related studies is presented in Table 13.

TABLE 13 Summary of in vivo model characterization and the inhibition ofhuman vessel growth of the M24met tumors in the SCID-chimera modelEndpoints CD31 (% inhibition Protocol Parameters Compared to Day ofTumor Drug Dose Route Schedule Control) Study General notes MCF-7 nonena na na not quantified 19 Tumors implanted intradermially. Tested withand with out estradiol and collagen implant. Tumors grew slowly andexpressed little human CD31 M24met none na na na not quantified 19Tumors implanted intradermially. Tested with and with out collagen/FNmatrix. With matrix found superior tumor growth, all future studies willcontain matrix supliments. Tumors showed good human CD31 staining M24metnone na na na not quantified 9 Tumor size <100 mm³. Little (small) humanCD31 M24met none na na na not quantified 12 Tumor size <100-200 mm³.(medium) Some human CD31 M24met none na na na not quantified 12 Tumorsize <200 mm³. (Large) Large M24met tumors have superior numbers ofhuman vessel staining, future studies will be conducted with largertumors M24met Non- 10 IV 2 doses 0 15 First screening study. Specificmg/kg (day 5 & 1.12.1(M29I/D19A) showed human 9) significant reductionof IgG human CD31 staining. No 1.12.1(M2 10 IV 42 tumor growthinhibition 9I/D19A) mg/kg observed M24met Non- 10 IV 2 doses 0 14 Secondscreening study. Specific mg/kg (day 5 & Confirmed results of GW- human10) 366. No tumor growth IgG inhibition observed 1.12.1(M2 10 IV 409I/D19A) mg/kg M24met Non- 10 SC 2 doses 0 14 First test of dosedependent Specific mg/kg (day 5 & activity of 1.12.1(M29I/ human 10)D19A) against human CD31. IgG Some dose dependent effect 1.12.1(M2 10 SC43 observed. PK results that 9I/D19A) mg/kg single dose will besufficient 1.12.1(M2 1 SC 50 for significant reduction in 9I/D19A) mg/kgCD31. No tumor growth 1.12.1(M2 0.1 SC 20 inhibiton observed 9I/D19A)mg/kg M24met No Dose 0 mg/kg na na

16 Second test of dose dependent activity of Isotype 10 SC one dose 01.12.1(M29I/D19A) against matched mg/kg (day 5) human CD31. Clear doseIgG dependent anti-CD31 effect Non- Specific human IgG 10 mg/kg SC

observed. No tumor growth inhibition observed 1.12.1(M2 1 SC 24 9I/D19A)mg/kg 1.12.1(M2 5 SC 59 9I/D19A) mg/kg 1.12.1(M2 10 SC 72 9I/D19A) mg/kgM24met Isotype 10 SC one dose 0 14 Final broad dose range test matchedmg/kg (day 5) for 1.12.1(M29I/D19A). IgG Study showed good dose1.12.1(M2 1 SC 33 dependent effects. Fitting 9I/D19A) mg/kg data to asigmoidal dose 1.12.1(M2 3 SC 41 response curve yields an 9I/D19A) mg/kgIC₅₀ of 93 nM. No tumor 1.12.1(M2 5 SC 60 growth inhibiton observed.9I/D19A) mg/kg 1.12.1(M2 7.5 SC 60 9I/D19A) mg/kg 1.12.1(M2 10 SC 739I/D19A) mg/kg 1.12.1(M2 50 SC 70 9I/D19A) mg/kg

Example 21 In Vivo EC₅₀ Determination

Human foreskin SCID chimera mice were intradermally implanted withM24met cells and were treated (sc) with anti-ALK-1 antibody1.12.1(M29I/D19A) at 1, 3, 5, 7.5, 10 and 50 mg/kg or with isotype matchanti-human KLH antibody (10 mg/kg). Upon the conclusion of theexperiment, human vessel area in each tumor were quantified as describedabove. Mouse plasma concentrations of anti-ALK-1 antibody1.12.1(M29I/D19A) were measured using the method described as following:serum samples from mice were analyzed for anti-ALK-1 antibody1.12.1(M29I/D19A) concentration by an ELISA (enzyme-linked immunosorbentAssay). ELISA plates were coated with 10 ug/ml goat anti-human IgG Fcspecific antibody (Pierce, cat# 31123) in PBS, incubated overnight at 4°C., and then blocked with StartBlock blocking buffer (Pierce, cat#37542)at room temperature for 1hr. Serum samples were diluted prior to theanalysis 100 and 1000-fold in StartBlock blocking buffer. Two sets ofstandards were prepared in the blank serum diluted 100 and 100-fold.Standards and diluted serum samples were incubated on the plate for 1hr.Bound anti-ALK-1 antibody 1.12.1(M29I/D19A) was detected usinghorseradish peroxidase (HRP)-labeled goat anti-human IgG (Fab-specific)antibody (Sigma, cat#A0293). The substrate used was 3, 3′, 5,5′-tetramethyl benzidine (Sigma, cat#T8665). Absorbance was read at 450nm on a Vmax plate reader (Molecular Devices, Menlo Park, Calif.). Astandard curve was fit using nonlinear regression. The detection limitof this assay was 10 ng/ml of anti-ALK-1 antibody 1.12.1(M29I/D19A).

SCID mouse plasma concentration of anti-ALK-1 antibody 1.12.1(M29I/D19A)is shown in FIG. 15.

FIG. 15 represents the estimated EC₅₀ for 1.12.1(M29I/D19A) in theM24met Foreskin SCID-chimera Model. Human vessel area was plottedagainst the average plasma PK across the study period (14 days) for eachtreatment group. A fitted curve was produced by the Sigmoidal DoseDependent program in the GRAPHPAD® (Prizm). EC₅₀ of 93 ng/ml (EC₅₀ isdefined as the plasma concentration required for a 50% reduction ofhuman vessel area in the Control group) was derived from the curve fit.

All publications, patents, and patent applications cited in thisspecification are incorporated herein by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

1. A monoclonal antibody, or an antigen-binding portion thereof, thatbinds activin receptor-like kinase-1 (ALK-1), comprising a firstvariable domain comprising SEQ ID NO: 6 and a second variable domaincomprising SEQ ID NO:
 8. 2. The monoclonal antibody of claim 1,comprising the heavy chain amino acid sequence of SEQ ID NO: 2 andcomprising the light chain amino acid sequence of SEQ ID NO:
 4. 3. Themonoclonal antibody of claim 1, comprising a heavy chain that comprisesSEQ ID NO: 6 and a light chain that comprises SEQ ID NO:
 8. 4. Themonoclonal antibody of claim 1 or 3, the monoclonal antibody beingselected from IgG1 or IgG2.
 5. A monoclonal antibody, or anantigen-binding portion thereof, that binds ALK-1, wherein said antibodyor antigen-binding portion comprises V_(H) CDR1, CDR2 and CDR3 sequencesin SEQ ID NO: 6 and V_(L) CDR1,CDR2 and CDR3 sequences in SEQ ID NO: 8.6. The antibody according to claim 1 that is an IgG, an IgM, an IgE, anIgA, or an IgD.
 7. A pharmaceutical composition comprising the antibodyor antigen-binding portion according to claim 1 and a physiologicallyacceptable carrier.
 8. A monoclonal antibody, or an antigen-bindingportion thereof, that binds ALK-1, the antibody comprising the V_(H)amino acid sequence encoded by the nucleotide sequence of the plasmidinsert found in the E. coli clone deposited under ATCC accession numberPTA-6864 and the V_(L) amino acid sequence encoded by the nucleotidesequence of the plasmid insert found in the E. coli clone depositedunder ATCC accession number PTA-6865.
 9. The antibody according to claim3 that is an IgG, an IgM, an IgE, an IgA, or an IgD.
 10. The antibodyaccording to claim 5 that is an IgG, an IgM, an IgE, an IgA, or an IgD.11. A pharmaceutical composition comprising the antibody according toclaim 2 and a physiologically acceptable carrier.
 12. A pharmaceuticalcomposition comprising the antibody according to claim 3 and aphysiologically acceptable carrier.
 13. A pharmaceutical compositioncomprising the antibody or antigen-binding portion according to claim 5and a physiologically acceptable carrier.
 14. A pharmaceuticalcomposition comprising the antibody or antigen-binding portion accordingto claim 8 and a physiologically acceptable carrier.
 15. A hybridomadeposited under an ATTC accession number of PTA-6808.